Equilibrium adsorption method for making a silica nanocarrier spion composition

ABSTRACT

Silica nanocarriers hybridized with superparamagnetic iron oxide nanoparticles (“SPIONs”) and curcumin through equilibrium or enforced adsorption technique. Methods for dual delivery of SPIONs and curcumin to a target for diagnosis or therapy, for example, for SPION-based magnetic resonance imaging or for targeted delivery of curcumin to a cell or tissue. The technique can be extend to co-precipitation of mixed metal oxide involving Ni, Mn, Co and Cu oxide. The calcination temperature can be varied from 500-900° C. The nanocombination is functionalized with chitosan, polyacrylic acid, PLGA or another agent to increase its biocompatibility in vivo.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a Continuation of U.S. application Ser. No.17/348,911, now allowed, having a filing date of Jun. 16, 2021, which isa Division of U.S. application Ser. No. 16/055,221, now U.S. Pat. No.11,471,542, having a filing date of Aug. 6, 2018.

BACKGROUND OF THE INVENTION Field of the Invention

The embodiments herein generally relate to the field of nanomedicine,molecular imaging, and drug delivery.

Description of Related Art

The “background” description provided herein is for the purpose ofgenerally presenting the context of the disclosure. Work of thepresently named inventor(s), to the extent it is described in thisbackground section, as well as aspects of the description which may nototherwise qualify as prior art at the time of filing, are neitherexpressly or impliedly admitted as prior art against the presentinvention.

Cancer Treatment Limitations. The scope of therapeutic approaches totreatment of deadly diseases such as cancer and diabetes as well asother metabolic disorders has been expanded and redefined by recentinterdisciplinary research between medicine and nanotechnology(nanomedicine). In particular, the treatment of cancer poses majorchallenges worldwide. Numbers of cancer-related deaths, morbidities, andincidence are all rising; there are over 6 million cancer related deathsworldwide. Cancer incidence is expected to continue to rise and thenumber of people with cancer is expected to rise to about 24 million by2035.

Cancer treatments, such as conventional surgery as well as chemo-,radio- and photodynamic-therapies, are insufficient to address thesechallenges. Further, an anticancer therapy for one type of cancer may beineffective for treatment of another type of cancer, for example, ananticancer therapy for skin cancer may be incompetent for treatingbreast, liver, lung, or colon cancer.

Conventional cancer treatments suffer from many other limitations. Whilesurgery is often an effective way to treat solid, non-metastaticcancers, additional therapy is frequently needed if residual ormetastatic cancer cells remain after surgery. Radiotherapy targets highenergy radiation to bodily locations containing cancer cells in order todamage and kill the cancer cells. However, in also exposes normal,non-cancerous cells to the radiation and can result in the formation ofsecondary malignancies. Chemotherapy is relatively non-selective and candamage normal cells and tissues as well as cancer cells. It often causesunacceptable side-effects especially when used over a long period oftime.

In view of these limitations of different conventional anti-cancertherapies, there is an imminent need for a way to selectively targetcancer cells in the body with less toxic agents that can selectivelyinhibit the growth of or kill cancer cells.

Curcumin and related curcuminoids may have such a potential if properlytargeted to cancer cells in amounts effective to inhibit cancer cellgrowth. However, curcumin exhibits difficult pharmacokinetic properties,such as poor solubility in aqueous media and poor bioavailability totissues containing cancer cells. Curcumin in an antioxidant found innature having a polyphenol structure and may exhibit anti-cancerpharmacodynamic properties. Many antioxidants are known to act againstcancers, including against leukemia, colon and lung cancer cells, andagainst other metabolic disorders through free radical scavengingabilities.

Curcumin is widely used as a traditional medicine. It has been longpostulated as an anti-cancer drug due to its antioxidant properties(Kant V, Gopal A, Pathak N N, Kumar P, Tandan S K, Kumar D. Antioxidantand anti-inflammatory potential of curcumin accelerated the cutaneouswound healing in streptozotocin-induced diabetic rats. IntImmunopharmacol. 2014 June; 20(2):322-30. doi:10.1016/j.intimp.2014.03.009. Epub 2014 Mar. 24. PubMed PMID: 24675438;Ak T, Güløin I. Antioxidant and radical scavenging properties ofcurcumin. Chem Biol Interact. 2008 Jul. 10; 174(1):27-37. doi:10.1016/j.cbi.2008.05.003. Epub 2008 May 7. PubMed PMID: 18547552).

Interestingly, it was also reported to induce reactive oxygen species(ROS) (Gersey Z C, Rodriguez G A, Barbarite E, Sanchez A, Walters W M,Ohaeto K C, Komotar R J, Graham R M. Curcumin decreases malignantcharacteristics of glioblastoma stem cells via induction of reactiveoxygen species. BMC Cancer. 2017 Feb. 4; 17(1):99. doi:10.1186/s12885-017-3058-2. PubMed PMID: 28160777; PubMed Central PMCID:PMC5292151; Larasati Y A, Yoneda-Kato N, Nakamae I, Yokoyama T, MeiyantoE, Kato J Y. Curcumin targets multiple enzymes involved in the ROSmetabolic pathway to suppress tumor cell growth. Sci Rep. 2018 Feb. 1;8(1):2039. doi: 10.1038/s41598-018-20179-6. PubMed PMID: 29391517;PubMed Central PMCID: PMC5794879).

When ROS are present at low levels, it induces tumor proliferation.However, when the ROS are at extremely high levels, it induces celldeath, which is one of the suggested anti-tumor mechanisms of curcumin(Gersey et al, 2017). Curcumin was reported to influence progressioninto the cell cycle (cyclin D1, p53), and affect anti- and pro-apoptoticpathways (Ravindran J, Prasad S, Aggarwal B B. Curcumin and cancercells: how many ways can curry kill tumor cells selectively? AAPS J.2009 September; 11(3):495-510. doi: 10.1208/s12248-009-9128-x. Epub 2009Jul. 10. Review. PubMed PMID: 19590964; PubMed Central PMCID:PMC2758121; Zeng et al, 2018; and Gersey et al, 2017). In hepatocellularcarcinoma cell lines, curcumin treatment resulted in a significantincrease in the cyclin-dependent kinase inhibitor 1A (CDKN1A), whichsubsequently resulted in cell cycle arrest (Zeng Y, Shen Z, Gu W, Wu M.Inhibition of hepatocellular carcinoma tumorigenesis by curcumin may beassociated with CDKN1A and CTGF. Gene. 2018 Feb. 2. pii:S0378-1119(18)30104-5. doi: 10.1016/j.gene.2018.01.083. [Epub ahead ofprint] PubMed PMID: 29408622). Curcumin treated glioblastoma tumorbiopsies had lower proliferation and reduced ability to form tumorcolonies (Gersey et al, 2017). These reports suggest that curcuminexhibits broad anti-tumor properties.

However, there are a numerous issues hindering the use of curcumin as atherapeutic drug. These issues include: rapid metabolism, poorabsorption, poor bioavailability, insolubility in water, and chemicalinstability. These problems may explain the conflicting results inclinical trials especially in that the administration was through theoral route (Nelson K M, Dahlin J L, Bisson J, Graham J, Pauli G F,Walters M A. The Essential Medicinal Chemistry of Curcumin. J Med Chem.2017 Mar. 9; 60(5):1620-1637. doi: 10.1021/acs.jmedchem.6b00975. Epub2017 Jan. 11. Review. PubMed PMID: 28074653; PubMed Central PMCID:PMC5346970).

Many forms of nanosilicas are used in non-medical applications includingtheir use in the chemical and electronic fields, for example, as partsof or supports for chemical catalysts, or for use in magnetic,electronic, dielectric, optical, batteries and other relatedapplications. Nanosilicas are currently being investigated forbiomedical applications including for drug delivery. For example, apayload molecule may be encapsulated within a nanosilicate carrier toattempt to improve payload biocompatibility, stability andbiodegradability; J. T. Cole, N. B. Holland, Multifunctionalnanoparticles for use in theranostic applications, Drug Deliv. andTransl. Res. 5 (2015) 295-309. A nanosilica carrier may shield a payloadmolecule from degradation or elimination in body and might function toimprove the pharmacokinetic properties of some molecules.

Prior attempts to load and deliver curcuminoids to target cancer tissueshave not been able to deliver adequate amounts of curcumin to a targettissue or to follow the distribution of curcumin once administered.These problems spring in part from a lack of a method to provide adeliverable form of curcumin.

Recent research by the inventors has shown that structured silica suchas SBA-16 has a great capability to carry acidic type antioxidants suchas gallic acid; V. Ravinayagam, B. Rabindran Jermy, Studying the loadingeffect of acidic type antioxidant on amorphous silica nanoparticlecarriers, 19 (2017) 190. However, in contrast to gallic acid,antioxidants such as curcumin are difficult to work with and presentspecific problems. For example, while curcumin encapsulated surfactantcomplexes, hydrogels, liposomes have been studied methods for theirpreparation are rather complex, especially for large scaleimplementation. Moreover, such methods provide products having reducedstability or solubility in biological environments; R. S. Mulik, J.Monkkonen, R. O. Juvonen, K. R. Mahadik, A. R. Paradkar, Int. J. Pharm.398 (2010) 190-203.

Curcumin encapsulated into MCM-41 type mesoporous silica was shown toimprove the solubility, enhance drug release, and result in a highercellular delivery; S. Jambhrunkar, S. Karmakar, A. Popat, M. Yu, C. Yu,Mesoporous silica nanoparticles enhance the cytotoxicity of curcumin,RSC Adv.4 (2014) 709-712. However, rapid disintegration of curcumin andcytotoxicity towards normal and cancer cell lines were observed; Q. J.He, J. L. Shi, F. Chen, M. Zhu and L. X. Zhang, Biomaterials, 2010, 31,3335.

Mesotextured silicas such as hexagonal MCM-41, SBA-15 and cubic SBA-16,MCM-18 have been reported to function as drug delivery agents in invitro and in vivo; I. I. Slowing, J. L. Vivero-Escoto, C. W. Wu, V. S.Lin, Mesoporous silica nanoparticles as controlled release drug deliveryand gene transfection carriers, Adv Drug Deliv Rev.60 (2008) 1278-1288;S. Kwon, R. K Singh, R. A. Perez, E. A. About Neel, H-W. Kim, W.Chrzanowski, Silica based mesoporous nanoparticles for controlled drugdelivery, J Tissue Eng. 2013; 4: 2041731413503357. However, recentstudies have shown poor bioavailability of drugs targeted with silicananocarriers and that so far only 5% of a drug reaches targeted tumors.This is comparable to the amount of a conventional drug, which does notcontain a nanocarrier, delivered. Conventional nanocarriers also lack ameans to track and measure targeted delivery of drugs such as curcuminto target sites.

Multifunctional theranostic nanoparticles have been used for drugdelivery and tumor identification. These represent combinations oftherapeutic compounds with tumor imaging agents; M. Howell, C. Wang, A.Mahmoud, G. Hellermann, S. S. Mohapatra, S. Mohapatra, Dual-functiontheranostic nanoparticles for drug delivery and medical imagingcontrast: perspectives and challenges for use in lung diseases, DrugDeliv. and Transl. Res. 3(2013)352-363.

Magnetic drug targeting has been proposed. This involves design of adrug or imaging composition that can be magnetically guided to a target.Prior attempts have shown that incorporating a drug and a magnetic agentin a single composition was cumbersome and challenging. Such acomposition can suffer from toxicity due to incorporation of toxiclevels of magnetic particles or from issues of biocompatibility such asinduction of inflammatory responses in respiratory system. However, someFDA approved SPIONs have intrinsic magnetic properties that may have thepotential to be used in combination with anticancer drugs. Moreover,SPION-based compositions have the potential to be functionalized ontheir iron oxide surfaces; J. Estelrich, E. Escribano, J. Queralt, M. A.Busquets, Iron Oxide Nanoparticles for Magnetically-Guided andMagnetically-Responsive Drug Delivery, Int J Mol Sci. 16 (2015)8070-8101. Magnetic Fe₃O₄-based mesoporous silica such as SBA-15 (p6mm), mesocellular foams and fiber type of silica have been previouslyprepared; S. Huang, C. Li, Z. Cheng, Y. Fan, P. Yang, C. Zhang, K. Yang,J. Lin, Magnetic Fe ₃ O ₄ mesoporous silica composites for drug deliveryand bioadsorption, Journal of Colloid and Interface Science, 376 (2012)312-321; Carbohydrate Polymers 171 (2017) 259-266.

Magnetic mesoporous silica particles have been used in hyperthermiatherapy; Z. Tian, X. Yu, Z. Ruan, M. Zhu, Y. Zhu, N. Hanagata, Magneticmesoporous silica nanoparticles coated with thermo-responsive copolymerfor potential chemo- and magnetic hyperthermia therapy, Microporous andMesoporous Materials 256 (2018) 1-9.

Amine-functionalized iron oxide/SBA-16 nanocomposites have been used asdual imaging tools and were able to carry large protein moleculesincluding antibodies; H. H. P. Yiu, H-j Niu, E. Biermans, G.vanTendeloo, M. J. Rosseinsky, Designed Multifunctional Nanocompositesfor Biomedical Applications Adv. Functional Mater. 2010, 20, 1-11.

Curcumin loaded in the SPIONs and coated hyaluronic acid (fluorescentdye) has been used for MM as well as fluorescent imaging studies; D.Lachowicz, A. Szpak, K. E. Malek-Zietekc, M. Kepczynski, R. N. Mullerd,S. Laurent, M. Nowakowska, S. Zapotoczny. Biocompatible and fluorescentsuperparamagnetic iron oxide nanoparticles with superior magneticproperties coated with charged polysaccharide derivatives, Colloids andSurfaces B: Biointerfaces 150 (2017) 402-407.

Magnetic nanosilicas have been used as transfecting agents; F. Scherer,M. Anton, U. Schillinger, J. Henkel, C. Bergemann, A. Kruger, B.Gansbacher, C. Plank, Gene Ther. 2002, 9, 102), and immunoassay; B. Q.Sun, W. Z. Xie, G. S. Yi, D. P. Chen, Y. X. Zhou, J. Cheng, J. Immunol.Method. 2001, 249, 85. Magnetic nanosilica drug carriers can respond toexternal magnetic fields and thereby assist bioimaging, magnetictargeting agent to carry drug and delivery.

Some kinds of magnetic nanosilicas have been shown to have the potentialfor use in treatment of cancer; F. G. Xu, Q. Y. Ma, H. C. Sha, Crit.Rev. Ther. Drug Carr. Syst. 2007, 5, 445, M. W. Wilson, R. K. Kerlan, N.A. Fidleman, A. P. Venook, J. M. LaBerge, J. Koda, R. L. Gordon,Radiology, 2004, 230, 287. The 3D cage type of cubic SBA-16 has beenwidely used as catalyst and adsorbent in fine chemical processes. SBA-16has a 3D porous network which could provide extensive diffusional accessto some drug molecules.

Many obstacles must be overcome to attain a magnetic delivery systemthat delivers adequate amounts of curcuminoids to cancer cells in thehuman body. For example, a magnetic structured silica drug carrier forcurcumin must have a suitable pore size for uptake, transport andsubsequent release of curcumin at a target site as well as a suitablepore size to accommodate magnetic particles. These problems includesolving the burst release problem, finding a suitable pore size forSPION and curcumin loading, and developing a suitable method for makinga structured silica-SPION-curcumin composition.

Burst Release and Delivery to Target Site. Existing drug carrierscontain a single kind of pore. For example, Q10 silica is onedimensional, MCM-41 is two dimensional, SBA-16 is three dimensional.While these carriers are reported to carry different kinds of drugs,they suffer from the problem of “burst release”. Burst release resultsin premature or over-release of a drug during its transit to a targetsite in the body, such as a tumor site. Burst release results in wastageof the drug during transit, exposure of the blood or non-target tissuesto concentrations of the drug increasing the risk of side-effects, andthe delivery of suboptimal dosage of the drug to the target site.Moreover, burst release increases the cost of pharmacological therapy asmore drug and potentially a longer period of treatment is oftenrequired. A complementary problem to burst release is how to facilitatethe release of curcumin once it reaches the target. A new curcumincarrier needed to be developed that can increase the percentage ofcurcumin loading, transfer the curcumin to a target site withoutsubstantial release, and then release curcumin into or around targetcancer cells which often reside in a more acid environment than normal,non-cancerous cells.

SPION size and Curcumin Loading. The designing of magnetic and drugloading in a single entity is cumbersome and challenging and developingsuitable pore sized magnetic drug carrier having a suitable pore size toload, transport and release antioxidant molecules such as curcumin at atargeted site has not been tried. Formulation of agglomerations ofnanosilicas with suitable nanoclusters of Fe₂O₃ particles for thesefunctions has not been reported. Development of nanosupports thatfacilitate drug-delivery and that provide useful magnetic properties fortumor imaging application is needed. In preferred embodiments curcumincan be loaded in the range between 0, 5, 10, 15, 20, 25, 30, 35, 40, 45,50, 55, 60, 65, or 70 wt % at the maximum.

SPIONs Preparation Difficulties. The preparation of Fe₂O₃ in desirednanosizes (such as ≤30 nm) are current challenges in material chemistry.In order to control the particle size, several efforts have been madeusing several different techniques such as hot injection method,controlled thermal disintegration and sonochemistry, etc. The formationof nanoclusters of Fe₂O₃ over SiSBA-16, Q-10 silica and mesocellularfoam have not been reported.

To address these disadvantages of conventional drug carriers, theinventors sought to develop a new kind of SPIONs/nanocarriernanoformulation for drugs such as curcumin that would increase thepercentage of drug loading, for targeted drug delivery and tumor imagingcapability. The above mentioned shortcomings, disadvantages and problemsare addressed herein and which will be understood by reading andstudying the following specification.

BRIEF SUMMARY OF THE INVENTION

The inventors provide a new way to deliver curcumin that effectivelyreduces the viability of cancer cells (MCF7 cells). In addition, theyprovide curcumin-loaded mesocellular foam silica nanoparticlesimpregnated with Fe₂O₃ allowing these nanoparticles to have a dualfunctions as chemotherapeutic drug carriers and as magnetic nanomaterialfor imaging or targeting purposes.

The invention provides a composition which enhances and bioavailabilityof curcumin at target tissues and provides for imaging site-specificuptake of curcumin. Curcumin has poor pharmacokinetic properties as itis relatively insoluble in aqueous media including blood plasma andtissue fluids. Treatment methods using curcumin also suffer from a lackof an ability to track tissues that take up curcumin. The inventionprovides a way to traffic and deliver larger amounts of curcumin to atarget tissue by including it as a cargo or payload in or on ananosilica carrier, such as one based on SBA-16, Mesocellular foam,and/or Q-10 silica that also incorporates superparamagnetic iron oxidenanoparticles (“SPIONs”). The invention permits magnetic direction ofthe nanosilica-SPION-curcumin particles to target tissues such as thosecontaining cancer cells for curcumin delivery.

In a first embodiment, the invention is directed to a compositioncomprising a platform of one or more types of nanoporous structuredsilica, at least one kind of magnetic nanoparticles in an amount rangingfrom about 5, 10, 15, 20, 25 to about 30 wt % based on total weight ofthe composition, and at least one curcuminoid. The platform ofstructured silica includes one or more of SiSBA-16, Q-10 silica,mesocellular foam (MSU-foam, mesocellular silica foam), silicalite,mesosilicalite, mesoporous silica, amorphous silica, SiKIT-6, ULPFDU-12or SiMCM-41. The nanocarriers can also include high silica zeolitesinvolving small, medium or large pore zeolites including ZSM-5,mordenite, Beta, HY, ZSM-11, ZSM-12, ZSM-22, and ZSM-23. The nanocarriercan also include carbon based nanocarriers such as mesocarbon, grapheneoxide etc.

The composition of may contain magnetic particles, which are preferablysuperparamagnetic iron oxide nanoparticles (“SPIONS”) which arenon-toxic compared to other magnetic materials based on highconcentration of cobalt and nickel. The composition may contain SPIONSmade of magnetite Fe₃O₄ and/or its oxidized form maghemite or γ-Fe₂O₃.

The magnetic nanoparticles in the composition of the first embodimentabove may contain Fe₂O₃ or a mixture of NiFe₂O₄, CuFe₂O₄, MnFe₂O₄ orCoFe₂O₄ and have an average particle size ranging from about 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17 to about 18 nm when the structure silicais MSU-foam; about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 toabout 21 nm when the structure silica is SiSBA-16 or a mesoporoussilica; or about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24 or 25 nm when the structure silica is Q-10 or an amorphous silica.These ranges include all intermediate values and subranges.

The curcuminoid in the composition is preferably curcumin or a mixtureof curcuminoids containing curcumin. Curcuminoids include as curcumin aswell as demethoxycurcumin and bisdemethoxycurcumin and theirgeomentrical isomers and metabolites. Preferably, a curcuminoid isincorporated or associated into the composition through an equilibriumor enforced adsorption technique.

In some embodiments of the composition of embodiment 1, the magneticnanoparticles and/or curcuminoids and/or silica particles may be coated,covered with or otherwise incorporated into a polymer. Such polymersinclude so-called smart polymers such as pH- or temperature sensitivepolymers that degrade at a pH or temperature around a tissue sitecontaining cancer cells. In some embodiments, one or more components ofthe composition is functionalized with chitosan, polyacrylic acid, PLGAor another agent to increase its biocompatibility in vivo.

The composition as described above may also include a chemical orbiological targeting agent, such as an antibody or other ligand thatbinds to a tumor-associated antigen or tumor marker or to markerscharacteristic of a target cell or tissue or characteristic of, orspecific to, various types or subtypes of cancers, neoplasms or tumors.

The composition of the first embodiment above may exhibit a degree ofmagnetization (M, emu/g) as measured by vibrating sample magnetometry(VSM) greater than an otherwise identical composition wherein theplatform of structured silica consists of SiSBA-16, Q-10 silica,mesocellular foam, silicalite, mesosilicalite, SiKIT-6, ULPFDU-12 orSiMCM-41. Preferably, the composition will contain MSU-foam, SiSBA-16 ora mesoporous silica, or Q-10 or an amorphous silica rather thanSilicalite, SiKIT-6, ULPFDU-12 or SiMCM-41.

The composition of the first embodiment above may exhibit a percentageof cumulative curcuminoid release, in phosphate buffered saline (PBS) atpH 5.6 and 37° C. over 3 hours, greater than an otherwise identicalcomposition wherein the platform of structured silica consists ofSiSBA-16, Q-10 silica, mesocellular foam, silicalite, mesosilicalite,SiKIT-6, ULPFDU-12 or SiMCM-41. Representative values for cumulativerelease range from about at least 10, 15, 20, to about at least 25%.Preferably, the composition will contain MSU-foam, SiSBA-16 or amesoporous silica, or Q-10 or an amorphous silica rather thanSilicallite, SiKIT-6, ULPFDU-12 or SiMCM-41.

Another embodiment of the invention is a method for treating a cancer,neoplasm, or tumor comprising administering the composition described bythe embodiments above to a subject in need thereof. The composition maybe administered by any route that contacts it with the target tissue orcells. For example, it may be administered orally, intragastrically,intraintestinally, intraluminally, or rectally for cancers of thegastrointestinal tract. Other routes include but not limited to topical,oral or nasal (including by inhalation), vaginal, parenteral (includingtopical, subcutaneous, intramuscular and intravenous) administration.The composition may also be administered in situ into or around acancer, neoplasm, or tumor.

In some embodiments the composition will be magnetically guided to atarget in the body of the subject after administration. In otherembodiments, the composition may be functionalized, for example, with aligand that binds to a tumor-associated antigen to enable it toselectively bind and accumulate at tumor sites.

In other embodiments, once the composition has reached its target tissueor target cancer cells it may be used to induce hyperthermia byapplication of radiation that releases heat when it contacts themagnetic particles in the composition. Advantageously this heat mayinhibit cancer cell growth or serve to release curcumin and other drugs,when present in the composition, into or around the cancer cells, forexample, when a smart or temperature-sensitive polymer is used to coatthe curcumin or nanoparticles.

Another embodiment of the invention is a method for detecting cancer,neoplasm, or tumor cells that includes administering the composition ofembodiments above that contains at least one agent that binds to orotherwise targets cancer, neoplasm, or tumor cells to a subjectsuspected of having a cancer, neoplasm, or tumor, detecting the magneticnanoparticles using nuclear magnetic resonance (NMR) or X-ray imaging,selecting a subject in which the magnetic nanoparticles display andabnormal accumulation, localization or distribution compared to acontrol subject not having a cancer, neoplasm, or tumor; and treatingthe selected subject for the cancer, neoplasm, or tumor. This method mayinvolve impregnating nanostructured pore surfaces of the platform ofstructured silica with magnetic nanoparticles, calcining the impregnatedplatform of structured silica, and adsorbing the curcuminoid to thecalcined platform of structure silica and magnetic nanoparticles. Insome embodiments calcining occurs at or between 450, 500, 550, 600, 650,700, 750, 800, 850, 900 or 950° C. In this embodiment a platform ofstructured silica is at least one of MSU-foam, SiSBA-16 or a mesoporoussilica, or Q-10 or an amorphous silica; the magnetic nanoparticles areSPIONS. One or more curcuminoids, preferably curcumin or a mixturecontaining curcumin is used for this embodiment. In some embodiments thecurcuminoid comprises >0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95 or 100wt % curcumin and may be adsorbed through an equilibrium or enforcedadsorption technique.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

FIG. 1 . X-ray diffraction pattern of curcumin/10 wt % SPIONs loadedover different nanocarriers; (a) curcumin, (b) Q-10, (c) Si-MCM-41, (d)Si-SBA-16, (e) mesocellular foam, (f) Si-KIT-6, (g) ULPFDU-12 and (h)Silicalite, respectively.

FIGS. 2A-2B show the N₂ adsorption isotherm of parent and 10 wt % Feimpregnated Q-silica, Si-MCM-41, SiSBA-16, MSU-foam, and SiKIT-6 silica.

FIGS. 2C-2D show the pore volume and pore width of parent and 10 wt % Feimpregnated silica nanocarriers. The BET surface area and porestructure, including pore surface area, pore volume and average porediameter, of different nanocarriers are shown in Table 1.

FIG. 3 . The magnetic properties of 10 wt % SPIONs loaded over differentnanocarriers.

FIG. 4 . Drs UV spectra of 10 wt % SPIONs loaded over differentnanocarriers: (a) Q-10, (b) Si-MCM-41, (c) Si-SBA-16, (d) MSU-foam, (e)Si-KIT-6, (f) ULPFDU-12 and (g) Silicalite, respectively.

FIG. 5 shows the FTIR spectra of (a) Q-10 silica, (b) SPIONs/Q-10, (c)Curcumin, (d) Curcumin/Q-10, (e) Curcumin/SPIONs/Q-10, (f)Curcumin/SPIONs/MSU-foam and (g) Curcumin/SPIONs/SiSBA-16, respectively.The chemical structure of curcumin is shown at the bottom of the panel.

FIGS. 6A-6F. SEM micrographs based on comparative surface morphologicalfeatures of 10 wt % SPIONs loaded over magnetically active nanocarrier.FIG. 6A: Q-10 silica; FIG. 6B: SPIONs/Q-10; FIG. 6C: SiSBA-16; FIG. 6D:SPIONs/SiSBA-16; FIG. 6E: mesocellular foam and FIG. 6F:SPIONs/MSU-foam, respectively.

FIGS. 7A-7F. Magnified SEM micrographs based on comparative surfacemorphological features of 10 wt % SPIONs loaded over magnetically activenanocarrier. FIG. 7A: Q-10 silica; FIG. 7B: SPIONs/Q-10; FIG. 7C:SiSBA-16; FIG. 7D: SPIONs/SiSBA-16; FIG. 7E: MSU-foam; and FIG. 7F:SPIONs/MSU-foam, respectively.

FIGS. 8A-8F. TEM images and average size of SPIONs. FIG. 8A:Fe/Si-SBA-16; FIG. 8B: Fe/Si-MCM-41; FIG. 8C: Fe/Q-10; FIG. 8D:Fe/MSU-Foam; FIG. 8E: Fe/Silicate; and FIG. 8F: bar graph of averagesize measurement with standard deviation for each specimen. Ten or morethan ten particles were taken for size estimation and shown in the formof average size. Two ranges of particles were found: one small sized(FIG. 8F, blue/left bars) and the second large sized (green/right bars).The scale bars correspond to 100 nm.

FIG. 9 . Pictorial representation of curcumin adsorption over SiSBA-16nanocarrier (a-e) and SPIONs/SiSBA-16 (f j) at 30-390 μg/mlcurcumin/nanocarrier in methanol-phosphate buffered saline (PBS) mixturestirred for 24 h.

FIGS. 10A-10D. Curcumin release profile in nanosilicas with, or withoutSPIONS, in PBS solution (pH 5) for 3 h. Nanocarrier supports (powderedform): MSU-foam and SiSBA-16 (FIG. 10A), Si-KIT-6 and ULPFDU-12 (FIG.10B), Q-10 and siMCM-41 (FIG. 10C) and Silicalite (FIG. 10D).

FIG. 11 . SPIONs/MSU-foam loaded with curcumin significantly reducescell viability. Percentage of cell viability with the followingtreatments: mesocellular foam silica (1^(st) set), Fe₂O₃ (2nd set),curcumin (3^(rd) set), mesocellular foam silica+Fe₂O₃ (4th set),MSU-foam silica+curcumin (5^(th) set), and MSU-foamsilica+Fe₂O₃+curcumin (6^(th) set). Treatment concentrations were 10,20, 40, 80, and 100 μg/ml for 24 h (n=5 independent experiments). Dashedline represents control, which is set as 100% cell viability. Errorbars, ±SEM. *P<0.05; **P<0.01 versus control. This study is in progress.

FIG. 12A. Graphic depiction of magnetically inactive SPIONS/SiMCM-41 andmagnetically active SPIONS/SiSBA-16 and SPIONS/MSU-foam. Schematicrepresentation shows the nature of SPIONs deposition over differentstructured silicas.

FIG. 12B. Cumulative release profiles of curcumin-loaded SPION/silicananoparticles versus magnetization.

DETAILED DESCRIPTION OF THE INVENTION

Structured Silicas. The structured silicas are tested as nanocarriers inbiomedical applications such as targeted oriented drug therapy,diagnostic purpose, stem cell and bioengineering. Mesoporous silicates,such as MCM-41 and SBA-15 are porous silicates with huge surface areas(normally ≥1,000 m²/g), large pore sizes (2 nm≤size≤20 nm) and orderedarrays of cylindrical mesopores with very regular pore morphology. Othermesotextured silicas include cubic SBA-16 and MCM-18. In someembodiments microstructured, mesoporous or macroporous silicas ormixtures thereof may be used. Most microporous silicas have average porediameters of less than 2, 1.75, 1.5, 1.25, 1, 0.75, 0.5 or 0.25 nm. Mostmesoporous silicas will have average pore diameters ranging from ≥2, 3,4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nm. Mostmacroporous silicas will have average pore diameters of >50, 60, 70, 80,90 or 100 nm.

The possibility of silane functionalization of structured silicas orfunctionalization with chitosan has led the applications to expandbeyond catalysis in fine chemical synthesis to magnetic, optical,battery and dielectric applications.

Nanocarriers used in the invention include (i) Q-10, (ii) SiSBA-16,(iii) mesocellular foam (MSU-F or MSU-foam), (iv) SiMCM-41, (v)ULPFDU-12, (vi) SiKIT-6 and (vii) silicalite. The structured silica canalso be derived from micro-meso Silicalite/SiMCM-41, or different highratio zeolite based composites. A zeolite can be ZSM-5, beta, USY,ZSM-11, silicalite, or other similar compounds. Other mesoporous silicamaterials having pore diameters between 2, 5, 10, 15, 20, 25, 30, 35,40, 45 and 50 nm may also be used in some embodiments of the invention.

Nanoparticles generally refer to particles having average diametersranging from about 1 to 100 nm, for example, 1, 2, 5, 10, 20, 50, <100or 100 nm (or any intermediate value or subrange thereof). In someembodiments, the nanoparticles of the invention will have averagediameters less than 50, 40, 30, 20, 10 or 5 nm. Average diameters may bemeasured by methods known in the art including by scanning electronmicroscopy (“SEM”).

SPION or Superparamagnetic iron oxide nanoparticles. SPIONs are composedof magnetite or iron oxide which is degradable in the body and non-toxiccompared to other magnetic materials such as cobalt and nickel. The mainforms of magnetite are Fe₃O₄ and its oxidized form maghemite or γ-Fe₂O₃.

SPIONs may be produced by methods known in the art, for example, asdescribed by Sun et al., J, American Chemical Society, 2002, 124, 8204.SPIONs may comprise one or more coatings or may be incorporated intomicelles or liposomes to enhance desirably pharmacokinetic propertiesincluding biological half-life, biocompatibility, and targeting. Thecompositions of the invention contain SPIONs of a size compatible within vivo administration and desired targeting functionality. Somerepresentative SPION particle sizes range from about 1, 2, 5, 10, 20,30, 40, 50, or 60 nm. A composition of the invention may contain asingle size or single size distribution of SPIONs or may contain two ormore sizes or size distributions. For example, various mixtures of largeSPIONs ranging from about 10 to 60 nm in average size and small SPIONsranging in size from about 2 to 22 nm may be used as described in Table1-2. Mixtures of SPIONs of different sizes permit tuning of a biologicalresponses or imaging functions. In some embodiments, a coprecipitationtechnique can be followed to form metal oxide composite with Ni or Cu orMn and Co nanoparticle to form respective MFe₂O₄ to enhance imagingcapacity by increasing magnetization property.

In some embodiments the core of the SPIONs may be magnetite which iscovered with one or more shells, for example, a polymer shell or a goldor metal shell. SPIONs may also be incorporated into, or coated with,one or more polymers including smart, pH-sensitive, ortemperature-sensitive polymers.

Functionalized super paramagnetic iron oxide nanoparticles (SPIONs) maybe used in accordance with one or more embodiments of the invention, forexample, a SPION (or other components of a composition of the invention)may be functionalized with one or more curcuminoids, or with acombination of one or more curcuminoids and a targeting ligand such asan antibody that binds to a tumor-associated antigen. In someembodiments SPIONs may be conjugated to targeting moieties such asligands that bind to, or agents that are internalized by, tumor orcancer cells or by other target molecules, receptors, cells or tissues.

The content of SPIONS, porous silica, and curcumin in a compositionaccording to the invention may be selected based on its intended use.However, some general content ranges for these components include fromabout 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19to about 20 wt % SPIONS, from about 80, 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 95 to about 95 wt % porous silica, and a curcuminadsorption to the silica-based nanocarrier of 10, 15, 20, 50, 100, 120,150, 200, 210, 220, 250, 300, 310, 320, 330, 340, 350, 360, 370, 380,390, 400, 420, to about 450 μg/ml curcuminoid. These ranges are based onadsorption in PBS containing 10 wt % methanol after 24 hr and includeall intermediate values and subranges.

In embodiments for use in vivo, curcuminoid adsorption to a nanocarriermay be performed in a medium not containing methanol which can be toxicin vivo. For example, a solvent such as acetone, ethanol, DMSO anddiemethylformamide (or a nontoxic or pharmaceutically acceptable organicor aqueous solvent) may be used in place of methanol. For particularapplications, an amount of curcumin or other curcuminoid may be selectedthat when administered in vivo inhibits the activity of histonedeacetylases: HDAC1, HDAC3, HDAC8, transcriptional co-activator proteinssuch as p300 histone acetyltransferase, or arachidonate 5-lipoxygenaseby at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or >90%.

Curcumin has the following structure:

A curcuminoid is a linear diarylheptanoid. This class of compoundsincludes curcumin in both its keto and enolate forms as well as curcuminderivatives such as demethoxycurcumin and bisdemethoxycurcumin and theirgeomentrical isomers and metabolites including sulfate conjugates andglucoronides. Other examples of curcumin derivatives or analogs includethose described by Raja, et al. U.S. Pat. No. 9,447,023 B2, Raja, etal., U.S. Pat. No. 9,650,404 B2, Johnson, et al., U.S. Pat. No.9,556,105 B2 or Vander Jagt, et al., U.S. Pat. No. 9,187,397 B2 (allincorporated by reference); especially for their descriptions ofcurcuminoid formulas and various chemical species of curcuminoids.

Mixtures of curcuminoids are also contemplated such as one isolated fromrhizomes of turmeric comprised of Curcumin (75-81%), Demethoxycurcumin(15-19%) and Bisdemethoxycurcumin (2.5-6.5%). The content of any one ofa curcuminoid in a mixture may range from about 0 to about 100 wt %, forexample, 10-90 wt %, 20-80 wt %, 30-70 wt %, 40-60 wt %, 50 wt %, 40 wt%, 33.3 wt %, 30 wt %, 20 wt %, 10 wt % or 5 wt % or 1 wt %. A mixturemay contain two, three or more different curcuminoids.

Curcumin may be present in a crystalline or amorphous form or in amixture of both crystalline and amorphous forms, for example at a ratioof 1-99 wt %:99-1 wt %, 10-90 wt %:90-10 wt %; 20-80 wt %:80-20 wt %,30-70 wt %:70-30 wt %, 40-60 wt %:60-40 wt % or about 50 wt %:about 50wt % (or any intermediate ratio of crystalline: amorphous forms. In someembodiments disclosed herein, curcumin will be in an amorphous form toincrease its solubility.

Curcumin and its derivatives are known for their antimicrobial,anti-oxidative, anti-inflammatory, and anti-cancer properties such asmalignancies in the brain or nervous system. Curcumin has also beenproposed as an agent to treat oxidative stress, such as oxidative stressin the brain, and for treatment of neurodegenerative disease likeAlzheimer's disease (“AD”) or Parkinson's disease (“PD”); Lee, et al.,Curr. Neuropharmacol. 2013 July; 11(4):338-378 (incorporated byreference).

Curcumin may also be functionalized or prepared as a conjugate withanother moiety to modify or improve its pharmacokinetic properties. Forexample, curcumin can be adsorbed through functionalization to a silane,carboxylic acid, or biotin. Moreover, biocompatibility ofcurcumin/SPIONs/mesosilica nanoformation can be increased by themodification with chitosan, or poly (D,L-lactide-co-glycolide), orpolyethylene glycol.

Smart Polymers. These represent a combination of nano- or micro-sizedsolid functional materials with one or more polymers. For example, amagnetic material, such as a SPION, may be incorporated or dispersedinto a polymer composite. Anisotropic properties may be conferred on thecomposite structure or particles by application of a magnetic fieldduring crosslinking or condensation of the polymer. In some embodimentsof the invention, SPIONs will be incorporated into, or covered with, apolymer to form nanoparticles with a polymer coating that can shield thebody from direct exposure to the SPIONs or control the rate of exposureand subsequent elimination of SPIONs. These nanoparticles may beproduced with anisotropic magnetic properties. In some embodiments,smart polymer coatings can be pre-applied to curcumin before or duringloading. In other embodiments smart polymers can cover the SPIONs andsilica nanoparticles.

In other embodiments, a curcuminoid or curcuminoid particles may beincorporated into, or covered with, a smart polymer that provides forcontrolled release of the curcuminoid. Smart polymer matrices releasedrugs by a chemical or physiological structure-altering reaction, oftena hydrolysis reaction resulting in cleavage of bonds and release of drugas the matrix breaks down into biodegradable components. While naturalpolymers may be used, artificially synthesized polymers such aspolyanhydrides, polyesters, polyacrylic acids, poly(methylmethacrylates), and polyurethanes may be used as well as conventionalpH- and temperature-sensitive polymers and copolymers.

A pH-sensitive polymer may be chosen to encapsulate or cover acurcuminoid, SPIONs or silica particles, and dissolve at a pH ortemperature around a tumor, and preferentially release the curcuminoidin or around a tumor, for example, in a tumor that is present in anacidic microenvironment. One example of a pH-sensitive polymer would bea polymer than degrades faster in a more acidic environment around acancer cell than at a pH around non-cancerous cells. Such polymers maybe selected depending on the type of cancer cell, its location and themetabolic status of the patient so that a curcuminoid will bepreferentially released in the relatively more acidic environment aroundthe cancer cell or under other conditions around or applied aroundtarget cells or in target tissues. Representative pH for tumormicroenvironments include 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6,6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, or <7.6. These generallylower pH or more acidic pHs are attributed to glycolytic activity andlactate release or to more rapid division by cancer cells. Themicroenvironments around many non-cancerous cells will be higher thanthose around cancer cells and may fall within a range of about 7.2, 7.3,7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0.

Hydrophilic, amorphous, low-molecular-weight polymers containingheteroatoms (i.e., atoms other than carbon) may provide for fasterdegradation. The rate of degradation may be controlled by adjusting thecomposition or thickness of the smart polymer to control the rate ofrelease of curcuminoid at a target site. Smart polymers have beendeveloped and are shown to respond to the external magnetic field aswell to pH and temperature changes; pH- and thermal-responsive magneticmicrocarriers for curcumin are described by. E. A. M. S. Almeida, I. C.Bellettini, F. P. Garcia, M. T. Farinácio, C. V. Nakamura, A. F. Rubira,A. F. Martins, E. C. Muniz, Curcumin-loaded dual pH- andthermo-responsive magnetic microcarriers based on pectin maleate fordrug delivery; Carbohydrate Polymers 171 (2017) 259-266. Smart polymersalso are described by, and incorporated by reference to, Filipcsei, etal., Adv. Polymer Sci. 206(1):137-189 (2007).

In some embodiments, the curcuminoid will be directly adsorbed, ornon-covalently or covalently associated with the nanoporous silicaand/or SPIONS. In other embodiments, the curcumin can be adsorbed orbound to the nanocarrier or SPIONs through functionalization of one ormore components of the composition, such as by functionalization ofcurcumin, silica or SPIONS with silanes, carboxylic acid, orbiotin/strepavidin. In other embodiments, the biocompatibility ofcurcumin/SPIONs/mesosilica nanocomposition can be increased by thefunctionalization of components of the composition with chitosan, orpoly (D,L-lactide-co-glycolide), or polyethylene glycol.

Methods and agents suitable for functionalization of SPIONS and othernanoparticles are described by Rimonidini,http://_www.cost-newgen.org/wp-content/uploads/2015/12/23-Sofia-COST-2015-rimondini.pdf(incorporated by reference, last accessed Apr. 20, 2018) and by Lee, etal., J Nucl Med 2013; 54:1-7 DOI: 10.2967/jnumed.113.122267(incorporated by reference), and Mishra, et al., Adv. Sci. 2017, 4,1600279, DOI: 10.1002/advs.201600279 (incorporated by reference).

Targeting to tumor antigens. Ligands such as antibodies that recognizetumor-associated antigens (or molecules such as receptors expressed athigher than normal levels by cancer cells) may be used to functionalizethe compositions of the invention. Tumor-associated antigens includeoncofetal antigens, such as alphafetoprotein (AFP, associated with germcell cancers or hepatocellular cancer) or carcinoembryonic antigen (CEA,associated with bowel cancer); tumor antigens such as CA-125 (ovariancancer), MUC-1 (breast cancer), epithelial tumor antigen (ETA,associated with breast cancer), tyrosinase (associated with malignantmelanoma), abnormal ras, p53 tumor antigens; abnormal proteins made byoncoviruses such as EBV or HPV; or abnormal cancer-associatedglycoproteins or glycolipids. Ligands that bind to tumor-associatedantigens may be conjugated to one or more elements of the composition ofthe invention, for example, to an iron oxide surfaces of SPIONS, bymethods known in the art such as with a cleavable or non-cleavablelinker, by tagging an element of the composition with biotin or(strep)avidin and the ligand with (strep)avidin or biotin, or bychemical conjugation

Magnetic targeting of drugs is known in the art and is incorporated byreference to Chertok, et al., Biomaterials 29(4), February 2008, Pages487-496 (brain cancer), Marcu, et al., Applied Surface Science 281, 15Sep. 2013, Pages 60-65 (breast cancer), and Dames, et al., NatureNanotechnology 2: 495-499 (2004) (lungs), each of which is incorporatedby reference. The composition of the invention advantageously is used totarget a curcuminoid to a cancer, neoplasm, or tumor. It may also beused to target the curcuminoid to other tissues including those of theenteral, urinary, or respiratory systems. In some embodiments, thecomposition of the invention will be magnetically targeted to a cancersite and the release of curcumin will also be controlled under theinfluence of a magnetic field. In some embodiments of the invention abiopolymer is used to improve SPIONs biocompatibility, targeted drugdelivery capability, magnetically active for magnetic resonance imaging(MRI). In other embodiments, a biopolymer release drugs in apH-dependent manner once magnetically localized to a tissue containingcancer cells.

Hyperthermia and hyperthermic treatment refer to subjection of a body ora portion thereof, to temperatures above 37° C., such as to temperatureof 40° C. or more, including 41° C. or more, such as 42° C. or more,such as 40 to 45° C., for a desired amount of time, e.g., 1 min orlonger, e.g., 5 min or longer, including 10 minute or longer, e.g., 1minute to 2 hours, such as 5 minutes to 1 hour.

Inductive hyperthermia. Devices or methods useful for inductivehyperthermia are known. These methods may use current magnetic fields incombination with ferromagnetic nanoparticles such as SPIONs. Devices andmethods for inducing hyperthermia are described by Kuroda, et al., Med.Biol. Eng. Comp. 37(3):2850290 (1999), by Araya, et al.,OncoTargetsTher. 6:237-42 (2013), or by Zhao, et al., Rare Metals, vol.25, issue 6, suppl 1, pp 621-625 (2006); which are incorporated byreference.

An anti-cancer agent (or anti-neoplastic agent or anti-tumor agent)encompasses all agents and therapeutics modalities known to one of skillin the art to ameliorate the symptoms in some manner of a cancer,neoplasm, or tumor. These include any agents, used alone or incombination with other agents or compounds, can reduce, ameliorate,trigger a state of remission of symptoms or markers associated withcancers, tumors, and the like, and can be used in methods andcompositions provided herein.

A chemotherapeutic agent includes any material or compound used in theart for the treatment of cancer. Chemotherapy can be conducted with alarge variety of agents and can include treatments with cisplatin,cisplatin-based compounds, carboplatin, mitomycin, vincristine,methotrexate, fluorouracil, calcium folinate, cytosine arabinoside,cyclophosphamide, epirubicin, etoposide, bleomycin A5, taxanes,mitoxanthrone, cylcophosphamide, topoisomerase inhibitors, angiogenesisinhibitors, cisplatin-based therapies, differentiation agents, signaltransduction inhibitors, busulfan, doxorubicin rapid dissolution,etoposide, 5-fluorouracil, tamoxifen, their salts, and combinationsthereof. Some embodiments of the invention will include compositionscontaining one or more chemotherapeutic agents in combination withcurcumin or a curcumin derivative.

One or more anti-cancer or chemotherapeutic agents may be used inconjunction with a composition according to the invention. It may beadministered before, simultaneously, or after the composition of theinvention or may be incorporated into a composition of the inventionalong with a curcuminoid. It may also be coadministered in a compositionsimilar to the invention where the curcuminoid is replaced by one ormore anti-cancer agents.

Cancer refers to a general term for diseases caused by any type oftumor, including solid tumors or tumors of the blood, and neoplasms. Asused herein, neoplasm refers to abnormal new growth, and thus means thesame as tumor, which may be benign or malignant.

Treatment describes at least an amelioration of one or more symptomsassociated with the condition afflicting the host is achieved, whereamelioration is used in a broad sense to refer to at least a reductionin the magnitude of a parameter, e.g. symptom, associated with thecondition being treated. As such, treatment also includes situationswhere the pathological condition, or at least symptoms associatedtherewith, are completely inhibited, e.g., prevented from happening, orstopped, e.g. terminated, such that the host no longer suffers from theside-effects or symptomatic side-effects of a treatment.

An anti-cancer treatment refers to any treatment designed to treat thecancer, tumor, or neoplasm by lessening or ameliorating its symptomsincluding its growth rate, ability to enter the circulatory system orlymph nodes, or to metastasize. Treatments that prevent the occurrenceof cancer, tumor, or neoplasm or lessen its severity are alsocontemplated.

EXAMPLES

The inventors show in the following Examples that nanoporous silicahybridized to magnetic nanoparticles (SPIONs) and loaded withcurcumin/SPIONs may be used for a dual purpose of drug delivery ofcurcumin and magnetic resonance imaging. As disclosed or shown by thefollowing Examples, the inventors have developed multifunctionalsupermagnetic iron oxide nanoparticles (SPIONs) based structured silicasuch as spherical silica, SiSBA-16 and mesocellular foam, to provide aneffective dual targeting magnetic nanomaterial for antioxidant(curcumin) delivery. A structured silica platform containing 10 wt %SPIONs was developed and provides an acquisition effect of curcumin in arange of about 30-390 μg/.

The effect of functionalization using different chain length of silanesand biocompatibility using chitosan fabrication were tested forcontrolled drug delivery.

Fe nanoparticles are incorporated into the structured silica andsilicalite through an enforced adsorption method.

The morphological variation of developed hybrid drug were scrutinizedusing various physico-chemical techniques such as X-ray diffraction(XRD), surface area analysis (BET), FTIR, Scanning electron microscope(SEM) and Transmission electron microscope (TEM).

The drug loading and delivery at various times were studied usingUV-Visible spectroscopy analysis. To investigate the cell viabilityeffects, curcumin-loaded/Fe₂O₃ impregnated mesocellular foam silicananoparticles which showed a high curcumin release effect were contactedwith MCF7 cells in vitro to assess cell viability using the MTT assay.

Example 1 Nanoporous Silica Platforms and Silica-SPION-CurcuminCompositions

Various kinds of nanoporous silica platforms, namely Q-10, Si-MCM-41,Si-SBA-16, MSU cellular foam, Si-KIT-6, ULPFDU-12 and silicalite, werehybridized with 10 wt % Fe SPIONs to form magnetically responsive silicathrough enforced adsorption technique. Curcumin was loaded into or ontothe magnetic silica through an equilibrium adsorption technique.

The phase, textural and morphological variation of developedmagnetically responsive silica and curcumin functionalization wasscrutinized using X-ray diffraction (XRD), surface area analysis (BET),Fourier transformed infrared spectroscopy (FT-IR), Scanning electronmicroscope (SEM) and Transmission electron microscope (TEM). Thecoordination of iron oxide over silica was studied using DRS-UVspectroscopy. The magnetization property was analyzed using VibratingSample Magnetometry (VSM). SPIONs loaded over Q-10, SBA-16 and MSU-Foamwere found to be magnetically active, while Si-MCM-41, Si-KIT-6 andsilicate were found to be magnetically inactive. 30-390 μg/ml ofcurcumin was loaded in 10% methanol in Phosphate buffered saline (PBS)mixture and the release study was carried out in PBS solution (pH 5.6)for 72 h at 37° C. The adsorption study shows that curcumin adsorptionover SPIONs hybrid silica (Q-10 and SiSBA-16) was boosted and was notaffected by iron oxide impregnation. The curcumin release study wascompared in the absence and presence of SPIONs over silica.

These Examples shows that drug release sustained in the presence ofSPIONs over silica than silica alone. Fe₂O₃ over MSU-Foam showed ahighest percentage cumulative release of 53.2% for 72 h, while SiSBA-16and Q-10 showed steady release with over 16% and 12% over 72 h,respectively. Further details of these Examples are provided below.

Experimental Section. The silica specified as CARiACT Q-10 with porediameter of 18.6 nm was purchased from Fuji Silysia Chemical Ltd, whilefoam type mesosilica termed as (MSU-F) was obtained from Aldrich. Thedetailed synthesis procedure for the support Si-MCM-41, Si-SBA-16,Si-KIT-6, ULPFDU-12, and silicalite was provided in an earlier publishedarticle; V. Ravinayagam, B. Rabindran Jermy, Studying the loading effectof acidic type antioxidant on amorphous silica nanoparticle carriers, 19(2017) 190.

Fe loading over nanocarriers through enforced adsorption technique.

An enforced impregnation technique was used to impregnate Fe₃O₄ into thepores of structured silica. Before the impregnation procedure, in orderto improve the impregnation, drying and vacuum treatment was performedto remove the pre-adsorbed moistures inside the pores. Alternatively,Fe₃O₄ impregnation can be performed through wet impregnation or can beincipient wetness route or can be incorporated during synthesis itself.

10 wt % loading of Fe was established by adding 0.7235 g of iron nitratenonahydrate in 80 ml of water, followed by stirring till dissolution.Then 1.0 g (1,000 mg) of nanocarrier was subsequently added and stirredfor 24 h at room temperature. After stirring, the material was driedwithout filtration at 120° C. for 3 h and the recovered material wasfurther calcined at 500° C. for 2 h.

In an alternative process silica can be impregnated with metal oxidessuch as gold, titanium, nickel, copper, manganese or other metal/oxidenanoparticles to produce a composite having different diagnostic orother functional properties. For instance, the impregnation can beperformed through coimpregnation of the silica with SPIONs andadditional active metal oxides based on nickel. In other embodiments thecalcination temperature of the mixture of Fe₂O₃/mesosilica may beselected within the range of about 300-800° C.

Curcumin adsorption through equilibrium adsorption technique. Curcuminadsorption over different nanocarriers and Fe impregnated nanocarrierswas carried out through an equilibrium adsorption technique. 1.0 g(1,000 mg) of nanocarrier was taken and added in the solution containing200-1,500 μg/ml of curcumin in 10% methanol in Phosphate buffered saline(PBS) mixture and stirred for 24 h. Then the solution was filtered,dried at room temperature. The percentage adsorption was calculatedbased on the equation:Percentage of curcumin adsorption (%)=(Initial curcumin conc−Finalcurcumin conc)/Initial curcumin conc×100.

The final curcumin concentration was calculated based on the equation:Final curcumin concentration=(Final absorbance value×Initial curcuminconc)/Initial absorbance value.

These results showed the final concentration of adsorption to range from30-390 μg/ml of curcumin.

Curcumin release. A curcumin (drug) release study was carried out in PBSsolution (pH 5.6) at 37° C. Specifically, for drug release study, 30 mgof (390 μg/ml curcumin/nanocarrier) sample was taken and dissolved in 50ml of PBS (pH 5) solution in a conical flask. Then the temperature wasraised to 37° C. and gradually stirred at 200 rpm for the following drugrelease study. At certain period, 10 ml of solution was withdrawn andreplaced with equal volume of fresh PBS solution. Then the releaseamount was calculated based on the calibration curve at specifiedwavelength of 428 nm.

Characterization. The X-ray diffraction pattern for mesostructuredsilicas was analyzed using bench top Rigaku Multiplex system. Thetextural characteristics (surface area, pore volume and pore sizedistribution) were measured using an ASAP-2020 plus, accelerated surfacearea and porosimetry, Micromeritics, Norcross, GA, USA. The magneticmeasurements were conducted with LDJ Electronics Inc. Model 9600 VSM inan applied field of 10 kOe. The calcined Fe/silica samples were measuredusing 60 mm dia integrating sphere equipped UV-Vis (Ultraviolet visible)V-750 diffuse reflectance spectroscopy (JASCO). Fe/silica and curcuminmethyl and functional groups were identified using Fourier transforminfrared spectroscopy (Perkin Elmer) equipped with attenuated totalreflectance (ATR). The average size and the surface morphology of the assynthesized specimens were measured using transmission electronmicroscope (TEM, FEI, and Morgagni, Czech Republic) and scanningelectron microscope (FE-SEM, TESCAN FERA3). SEM was performed atoperating voltage of 20 kV and TEM at 80 kV. For SEM, the samples weremounted onto metallic stubs with a double-sided adhesive tape. Goldcoating of a few nanometers was applied on specimens using sputtercoating machine (Quorum, Q150R ES, UK) to avoid charging and capturehigh quality electronic micrographs. Low and high magnification SEMimaging was performed to capture the recognized features of thespecimens. TEM samples were prepared by dropping particle dispersionsonto carbon-coated Cu grids and air-dried before mounting into themicroscope. Particle sizes were measured from electronic images usingGatan digital micrograph software. The data is presented in the form ofaverage number for each specimen with a standard deviation.

The X-ray diffraction patterns of pure curcumin and curcumin adsorptionover 10 wt % SPIONs loaded over different nanocarriers are shown in FIG.1 : (a) pure curcumin, (b) Q-10, (c) Si-MCM-41, (d) Si-SBA-16, (e)Mesocellular foam, (f) Si-KIT-6, (g) ULPFDU-12 and (h) silicalite,respectively.

In the case of pure curcumin (a), various diffraction peaks over the 2theta range 15-30° were observed indicating characteristics crystallinephase of curcumin; S. Mutalik, N. A. Suthara, R. S. Managuli, P. K.Shetty, K. Avadhani, G. Kalthur, R. V. Kulkarni, R. Thomas, Developmentand performance evaluation of novel nanoparticles of a grafted copolymerloaded with curcumin, Int J BiolMacromol. 86 (2016) 709-720.

In contrast, curcumin loading over SPIONs/different structurednanocarriers showed no such crystalline peaks indicating effectivetransformation of curcumin into amorphous state. These data show thatapart from silicalite, such a noncrystalline state of curcumin wasachieved over all types of structured silica irrespective of theirstructural domains of one dimension (1D), two dimension (2D), andthree-dimension (3D).

Past work has attributed transformation of a crystalline drug to anoncrystalline amorphous form to the confinement of drug inside thegeometrically constructed nanopores; F. Wang, H. Hui, T. Barnes, C.Barnett, C. Prestidge, Mol. Pharm. 7 (2009) 227-236. In particular,cubic cage nanopores of SBA-16 were reported to be effective for suchcrystalline transformation of drug to nanoform. In case of carvedilolmolecules (CAR), the presence of cage type of 3D nanopores of SBA-16 wasreported to thwart the transformation of the CAR molecules intocrystalline state by preventing the extension of the crystal latticeinside the 3D nanopores; Hu, Z. Zhi, Q. Zhao, C. Wu, P. Zhao, H. Jiang,T. Jiang, S. Wang, 3D cubic mesoporous silica microsphere as a carrierfor poorly soluble drug carvedilol, Micropor. Mesopor. Mater., 147(2012) 94-101. Similarly, in the case of amorphous type of silicas, suchas (b) Q-10 silica and (c) SiMCM-41 a characteristic broad peak ofsilica at about 2 theta range of 22° was not observed indicating effectof SPIONs impregnation.

The presence of Fe₃O₄ was expected to be observed at 2 theta value of35.45°. However the XRD pattern of all SPIONs/nanocarriers showed nosuch peak, indicating weak and broadening of such peaks due to smallnanosized Fe₃O₄ particles, which are attributed to the lack ofcrystallization at such nanopores of nanocarrier, see FIGS. 1 (b)-(h).

In case of nanocarriers without SPIONS, typical isotherm patterns wereobserved. The mesocellular foam exhibited type IV isotherm due tocellular foam structure. In case of Si-SBA-16 and Si-KIT-6, H1 typeisotherm appears indicating typical cubic cage type pores. The Si-MCM-41exhibited reversible type IV isotherm pattern with uniform pore sizedistribution; see FIGS. 2A and 2C.

The textural changes in the absence, as shown in FIGS. 2A and 2C, and ofSPIONs, as shown in FIGS. 2B and 2D, were evaluated using N₂ adsorptionisotherm technique. FIGS. 2A-2B show the N₂ adsorption isotherm ofparent and 10 wt % Fe impregnated Q-10 silica, Si-MCM-41, SiSBA-16,mesocellular foam, and SiKIT-6 silica. FIGS. 2C and 2D show plot porevolume (cm³/g·nm) against pore width (nm). Table 1 below describes theBET surface area and pore structure of different nanocarriers.

TABLE 1 Textural Properties of parent and 10 wt % Fa impregnated overdifferent structured silica. Cumulative BET Pore Pore Average SurfaceSurface Volume Pore Fe/Nanocarrier area Area (cc/g) Diameter [wt %g⁻¹suport] [m²/g-support] ^(a) [cm³/g-support]^(b) [cm³/g-support]^(c)[nm]^(d) Q-10 (1D) 233 270 1.08 18.6 10 wt % Fe/Q-10 258 274 1.02 15.8Si-MCM-41 (2D) 942 1200 0.88 3.7 10 wt % Fe/Si-MCM-41 951 1022 0.71 3.0Si-SBA-16 (3D) 677 337 0.48 2.8 10 wt % Fe/Si-SBA-16 327 194 0.33 4.0MSU-Foam (3D) 525 554 2.27 40.2 10 wt % Fe/MSU-Foam 140 134 1.30 40.2ULPFDU-12 (3D) 270 284 0.33 4.7 10 wt % Fe/ULPFDU-12 9 7 0.02 13.1SI-KIT-6 (3D) 878 862 1.23 5.7 10 wt % Fe/Si-KIT-6 676 616 0.96 5.6 ^(a)BET surface area, ^(b)Pore surface area, ^(c)pore volume and ^(d)averagepore diameter measured using BJH isotherm.

In case of Q-10 silica, after impregnation, nonsignificant changes wereobserved with respect to both specific (258 m²/g) and cumulative surfacearea (274 m²/g), while appreciable pore filling of about 16% (1.22 to1.02 cm3/g) along with pore diameter decreases from 18.6 to 15.8 nm wasobserved (Table 1).

Similarly, the surface area of Fe₂O₃/Si-MCM-41 slightly increased from923 m²/g to 951 m²/g, while an 11.2% decrease in cumulative surfacearea, and a 19.3% decrease in the pore volume was observed. The porediameter only slightly varied from 3.1 to 3 nm after Fe₂O₃ deposition.

In case of Fe₂O₃/Q10, surface area deposition remains negligibly small,whereas significant pore volume and pore diameter variations occur. Inthe case of Fe₂O₃/Si-MCM-41, cumulative surface area and pore volumedecreases, while pore diameter remained unaffected. This shows Feimpregnation over Q10 fills the pore volume and eventually affects porediameter. In the case of 3D cubic SBA-16, a significant decrease in thetextural characteristics was observed. Specifically, a decrease ofspecific surface area from 980 m²/g to 327 m²/g, and cumulative surfacearea from 591 m²/g to 194 m²/g, which is about 67% of Fe occupation wasobserved after Fe impregnation. The cumulative pore volume showed asimilar decrease (32%) compared to parent SiSBA-16. Reversely, theaverage pore diameter increases from 3.3 nm to 4.0 nm. The analysisshows that both surface area and pore volume are being affected andbeing filled in the 3D pore structure, while enlargement of pore sizeshows deposition of Fe₃O₄ around the pore walls that helps to expand thepore size.

In the case of MSU-Foam, reversely a significant change was observedwith isotherm and capillary condensation, while pore volume remainsmostly unchanged. The pore diameter showed significant variation. Thetexture of foam type of silica before Fe impregnation was of mesoporoustype with high surface area of 554 m²/g, with large pore volume of 2.27cc/g. The average pore size diameter was of 16 nm before impregnation.The isotherm pattern of mesocellular foam (parent form) and after Feimpregnation are shown in FIGS. 2A and 2B.

Before impregnation, the foam showed characteristic type IV isothermpattern with H1 hysteresis loop indicating well distributed cells alongwith windows; P. Schmidt-Winkel, C. J. Glinka, G. D. Stucky,Microemulsion Templates for Mesoporous Silica, Langmuir 16 (2000)356-361. After impregnation, a significant textural change with respectto surface area and pore volume was observed. A shift in capillaryfilling P/P0 range are observed. Specifically, an occupation of about73% was observed leading to specific surface area reduction from 525m²/g to 140 m²/g and about 76% occupation (from 554 m²/g to 134 m²/g)with respect to cumulative surface area was observed. In the case ofpore shape retainment, 57.3% of pore filling was observed. The porediameter of cellular foam increases from 16.4 nm to 40.2 nm.Significantly, the pore diameter showed significant alteration after Feimpregnation. Compared to parent MSU, the pore diameter increases from16.4 nm to 40.2 nm. Such pattern shows external agglomeration of Fe₂O₃particles at the pore surface contributing to expansion in the poresizes.

The cage type of mesoporous with Fm3m structure (ULPFDU-12) showedtypical broad hysteresis indicating interrelated large pores with smallsized window type of pores. In this type of material, an abrupt loss inthe textural property was observed. With 10 wt % Fe impregnation, about91% surface occupation was observed, where decrease in surface areaoccurs from 270 m²/g to 9 m²/g. The pore volume reduced significantly ofabout 94% from 0.33 ccg⁻¹ to 0.02 ccg⁻¹. With respect to pore sizedistribution, similar to cellular foam type, pore size expansionoccurred with impregnation from 4.7 ccg⁻¹ to 13.1 ccg⁻¹.

The cubic structure of Si-KIT-6 with Ia3d symmetry showed 77% oftextural filling with 676 m2/g specific surface area and 71% with 616m2/g cumulative surface area occupation with Fe impregnation. UnlikeSi-SBA-16, the pore volume of KIT-6 was sufficient to accommodate theimpregnated iron oxide particles. As observed with Si-SBA-16 and MSUFoam type of silicas, the external pore agglomeration was not observed.The impregnation led to the pore volume occupation of 78% that reducesfrom 1.23 ccg⁻¹ to 0.96 ccg⁻. In addition, KIT-6 pore diameter onlymarginally reduces from 5.7 to 5.6 nm.

These data show that Si-MCM-41 provided more pore filling, followed bycubic type Si-SBA-16, while Q-10 and MSU-Foam type showed externaldeposition of Fe₂O₃ particles, while pore volume remains largelyunfilled.

The average size measurement of SPIONs with standard deviation for eachsample was calculated (Table 1-2). The average size of the first set ofparticles was in the range of 3-21 nm and the second of 13-58 nm. Theorder of SPIONs particle size was found to be in the following order:Silicalite>Q-10>Si-SBA-16>MSU-Foam>Si-MCM-41. Specifically, averageSPIONs particle size of the Fe/Si-SBA-16 measured from TEM images wasfound to be 21.0±1.1 and 9.0±0.3. In support Si-MCM-41, finely dispersedSPIONs in the range of 3-13 nm was observed. Among the support,silicalite showed larger particles in the range of 21-58 nm followed byQ10 silica, which showed of 10-25 nm (FIG. 6 ).

TABLE 1-2 SPIONs Average particle size estimation (ten or more than tenparticles are considered for average size estimation). Sample LargeSPIONs particle Small SPIONs particle Code Nanocarriers size (nm) size(nm) ND-53 Fe/SiSBA-16 21.0 ± 1.1 9.0 ± 0.3 ND-48 Fe/SiMCM-41 13.0 ± 1.13.0 ± 0.2 ND-49 Fe/Q-10 25.0 ± 1.1 10.0 ± 0.3  ND-51 Fe/MSU 18.0 ± 1.07.0 ± 0.3 ND-47 Fe/Silicate 58.0 ± 5.5 21.0 ± 1.5 

FIG. 3 shows the magnetic property of 10 wt % SPIONs loaded over thedifferent nanocarriers: Q-10, Si-MCM-41, Si-SBA-16, mesocellular foamand Si-KIT-6 Fe/Q-10, respectively (top-to-bottom). Among the differentnanocarriers, a magnetically active support order was determined asFe/Q-10<Fe/SBA-16<Fe/MSU-Foam<Fe/Si-MCM-41<Fe/SiKIT-6. These data showedthat Q-10 followed by Si-SBA-16 and MSU-Foam was active, while SiMCM-41and silicalite was not active.

In case of Fe/Si-KIT-6, though the pore structure was similar to that ofSi-SBA-16, did not showed positive magnetization for the dualapplication of magnetically driven drug delivery approach. The presenceof narrow hysteresis loop showed the superparamagnetic behavior ofFe/Q-10, Fe/Si-SBA-16 and Fe/MSU-Foam, respectively. It has beenreported that such paramagnetic Fe3⁺ ions are formed throughincorporation at the pore walls of support; N. I. Cuello, V. R. Elias,S. N. Mendieta, M. Longhi, M. E. Crivello, M. I. Oliva, G. A. Eimer,Materials Science and Engineering C 78 (2017) 674-681. In case ofsilicalite, in spite of large iron oxide particles deposition, showedweaker magnetization. These data showed that three samples namelyFe/Q-Fe/Si-SBA-16 and Fe/MSU-Foam has the reasonable intrinsicmagnetization capability that can be utilized in addition to drugdelivery.

FIG. 4 depicts 10 wt % SPIONs loaded over the different nanocarrier (a)Q-10, (b) Si-MCM-41, (c) Si-SBA-16, (d) MSU-foam, (e) Si-KIT-6, (f)ULPFDU-12 and (g) Silicalite, respectively; it also shows the Drs-UVspectra for SPIONs loaded on structured silica samples. These datashowed that in spite of loading similar amount of SPIONs, thecoordinative dispersion of nanoparticle varies depending on thestructural integrity of silicas such as spherical Q-10, hexagonalSi-MCM-41, cubic type of pores of Si-SBA-16, Si-KIT-6, ULPFDU-12, MSUFoam and silicalite. The presence of three types of bands with varyingdegree of intensity at about 250 nm, 370 nm and 500 nm was observed foranalyzed samples. The characteristic absorption band between 200-300 nmshows the dispersed Fe3⁺ cation in tetrahedral coordination due to dπ-pπcharge transfer (Fe—O). The band appearance between 300-450 nm shows theformation of small oligomeric nanocluster, while Fe₃O₄ larger clustersare indicated through the presence of broad band between 450-600 nm; Y.Wang, Q. Zhang, T. Shishido, K. Takehira, J. Catal. 209 (2002) 186-196.

In the case of Q-10 silica sample, in addition to tetrahedralcoordination, a broad peak appears and extends up to 600 nm; FIG. 4 ,line (a). Particularly, an intense peak absorption band are observed atabout 520 nm that shows formation of large nanoclusters. Therefore,large microspheres silica of Q-10 silica have been shown to assistformation of octahedral species due to extra-framework iron oxidespecies than isolated tetrahedral iron oxide species. The formation ofsuch octahedral coordinated species are reported to occur due tonanoclusters; N. Cuello, V. Elias, S. Urreta, M. Oliva, G. Eimer,Microstructure and magnetic properties of iron modified mesoporoussilica obtained by one step direct synthesis, Materials ResearchBulletin 48 (2013) 3559-3563.

In the case of hexagonal pore channels Si-MCM-41 containing SPIONs, thepresence of intense tetrahedral species at about 280 nm shows that finedispersion of iron oxides incorporated in to the framework throughSi—O—Fe linkage, see FIG. 4(b); Y. Lu, J. Zheng, J. Liu, J. Mu,Microporous Mesoporous Mater. 106 (2007) 28-34. The formation of smallnanoclusters to smaller extent was also observed with less intenseabsorption band at 460 nm. Overall, SPIONs/Si-MCM-41 shows formation offinely dispersed SPIONs nanoparticles. Such decreased absorption atlonger wavelength shows the systematic deposition and stabilization ofFe₂O₃ inside the mesopores leading to reduced mobility of suchnanospecies after calcination; N. Cuello, V. Elias, S. Urreta, M. Oliva,G. Eimer, Microstructure and magnetic properties of iron modifiedmesoporous silica obtained by one step direct synthesis, MaterialsResearch Bulletin 48 (2013) 3559-3563.

In case of SPIONs/SiSBA-16, in addition to tetrahedral species asignificant proportional of extra framework species occurs at about 530nm FIG. 4 (c). The less intense tetrahedral absorption band between200-300 nm shows that distribution of particles is not fine compared tothat of Si-MCM-41 support but rather agglomerated type similar to thatof Q-10 silica. In particular, the main reason for such agglomerationover cubic Im3m pores of SBA-16 could be attributed to restricted poreentrance size that are relatively smaller than primary mesopore thuslimiting the intraparticle mass transfer.

SPIONs/mesocellular foam showed three types of absorption bands, FIG. 4(d). The foam like pore structure of mesocellular silica induced anabsorption peak at 250 nm indicating the presence of tetrahedral Fespecies. Comparatively, an intense absorption peak at 370 nm for foamsilica showed the presence of small nanoclusters to larger extent, whilevisible absorption peak at 520 nm shows existence of some largenanoclusters.

Overall the presence of small sized agglomerated octahedral species wasfound to be higher than Si-MCM-41, while large type of nanoclusters arelesser than that found in SBA-16 cage type of pores and sphericalsilica. SPIONs/SiKIT-6 with cubic Ia3d pores showed a prominent isolatedtetrahedral and small nanoclusters, FIG. 4 (e), compared to cage type ofIm3m structure, which showed external agglomerated octahedral species.The large pore volume (1.23 cc/g) of Ia3d cage type of pores of Si-KIT-6showed the difference with respect to pore filling ability compared toSi-SBA-16 counterpart with Im3m structure (0.49 cc/g) (Table 1).

In addition, the presence of large pore size distributions of Si-KIT-6showed a non-significant change in pore diameter from 5.7 nm to 5.6 nm,while Si-SBA-16 showed an increase in pore size from 3.3 nm to 4.0 nmindicating pore expanding due to Fe₂O₃ deposition around the thick porewalls of Si-SBA-16.

In case of SPIONs/ULPFDU-12, the presence of broad peaks showscharacteristics of variable Fe₂O₃ deposition occurs at the externalsurface area; FIG. 4(f). However, structural irregularity with respectto specific surface area, and pore volume (Table 1) shows thedeteriorating impact of Fe₂O₃ impregnation inside the mesopores leadingto disintegration of overall structural parameters (Table 1). The SPIONsover Silicalite (MEI structure) showed no strong absorption bandscorresponding to tetrahedral or octahedral coordination; FIG. 4(g).

FIG. 5 shows the FTIR spectra of (a) Q-10 silica, (b) SPIONs/Q-10, (c)Curcumin, (d) Curcumin/Q-10, (e) Curcumin/SPIONs/Q-10, (f)Curcumin/SPIONs/mesocellular foam and (g) Curcumin/SPIONs/SiSBA-16,respectively. Consistent with earlier work (R. Bhandari, et al., Mater.Sci. Eng. C 67 (2016) 59-64), the silica (Q-10) and Fe₃O₄ impregnatedsilica (Q-10) sample did not reveal any significant details; FIGS. 5 (a)and (b).

Curcumin spectrum showed the vibration of free hydroxyl groups withdistinct peak at 3507 cm⁻¹, carbonyl group at 1625 cm⁻¹, carbonyl andcarbon-carbon double bond at 1603 and 1505 cm⁻¹, methylene (CH₂) bendingvibrations at 1455 cm⁻¹, and 1428 cm⁻¹, and several peaks correspondingto —C—O—C symmetric and asymmetric vibrations are observed between1000-1300 cm⁻¹; FIG. 5(c).

After curcumin loading over Q-10 silica, additional peakscharacteristics of curcumin were observed; FIG. 5 . Plot (d).

Bhandari et al. stated that magnetic nanoparticle alone has thecapability to hold curcumin through functionalization. Similarly, in thepresent case, FTIR spectra, FIGS. 5(e) and (g) show that curcumin hasbeen effectively functionalized over SPIONs/silica hybrid composite.

A distinct peak at 962 cm⁻¹ corresponding to the enolic hydroxyl group(>C═C(OH)—) was observed for curcumin. After loading curcumin overSPIONs/silica hybrid composite, the peak corresponding to such in-planebending of OH group of enol decreases considerably indicatingfunctionalization route of curcumin through keto enol functional group.The observed functionalization trend is line with Fe₃O₄ nanoparticle forcurcumin functionalization. In case of curcumin, the presence ofdistinct peak at 3504 cm⁻¹ shows the hydroxyl functional group. Suchband of peak with reduced intensity was also observed forcurcumin/SPIONs/structured silica samples.

Compared to curcumin, the peak corresponding to carbon-carbon doublebond and carbonyl group at 1603 cm⁻¹ reduced significantly indicatinginteraction of curcumin with SPIONs hybridized silica. It has been shownthat the intactness of peak at 1023 cm⁻¹ assigned to surfacefunctionalization of C—O—C stretching of C₆H₅—O—CH₃ group over Fe₃O₄nanoparticle; P. R. K. Mohan, G. Sreelakshmi, C. V. Muraleedharan, R.Joseph, Vib. Spectrosc. 62 (2012) 77-84. These data show that the peakcorresponding to C₆H₅—O—CH₃ group are seen for curcumin at 1025 cm⁻¹,but after functionalization, broadening of such peak over SPIONshybridized Q-10 silica and Si-SBA-16 indicates functionalization at thestructured nanopores; FIG. 5 , plots (e) and (f).

In case of MSU, the presence of small peak was clearly visible at 1028cm⁻¹, indicating presence of certain proportion of curcumin at theexternal surface; FIG. 5 (g).

FIGS. 6 and 7 show the SEM micrographs based on comparative surfacemorphological features of 10 wt % SPIONs loaded over magnetically activenanocarrier: Q-10 silica (FIGS. 6A/7A), SPIONs/Q-10 (FIGS. 6B/7B),SiSBA-16 (FIGS. 6C/7C), SPIONs/SiSBA-16 (FIGS. 6D/7D), mesocellular foam(FIGS. 6E/7E) and SPIONs/mesocellular foam (FIGS. 6F/7F), respectively.The magnification was set to an appropriate value in order to capturethe representative features of the specimens in each case.

The Q10 silica shows the presence of spherical shaped microspheres withestimated average size of 100 μm sizes. In case of SPIONs/Q-10, theregularity of the spheres was affected by non-crystalline Fe₃O₄ loadedthrough enforced impregnation technique followed by calcination (FIGS.6A and 6B). The deposition of nanoclusters was clearly seen at higherscale bar 50 μm compared to parent Q-10 silica (FIGS. 7A and 7B).

A similar irregularly shaped microsphere morphology but with lessaverage sized spheres (˜4 μm) was observed in case of SiSBA-16 andSPIONs/SiSBA-16 (FIGS. 6C and 6D).

In case of MSU-Foam, the lower scale bar shows the presence of irregularagglomerated silica forms are observed (FIGS. 7C and 7D). Compared toMSU-Foam (FIGS. 6E and 6F), SPIONs/mesocellular foam clearly shows theporous morphological characteristics changes with agglomeratednanospheres structures at lower scale bar of 3 μm (FIGS. 7E and 7F).

The samples morphology and structure were further analyzed by TEM. FIG.8 shows the TEM images of 10 wt % SPIONs loaded over differentnanocarrier (FIG. 8A) SiSBA-16, (FIG. 8B) Si-MCM-41, (FIG. 8C) Q-10silica, (FIG. 8D) MSU-Foam and (FIG. 8E) Silicate, respectively.

The TEM analysis shows that SPIONs deposition are unique and depends onthe support nature, where the dispersion and agglomeration vary based onthe nanocarriers pore architecture. For instance, with three dimensionalcage type of SBA-16 pores, the presence of agglomerated forms of SPIONsas nanoclusters were observed along the pore channels (FIG. 8A), whilein hexagonal Si-MCM-41 support, the Fe₂O₃ particles are finely dispersed(FIG. 8B). The cage type of porous layer of SBA-16 appears to behomogeneous with a fairly constant thickness, and particles were foundconnected to the layers.

In the case of microsphere Q10 silica, MSU-Foam and silicalite, externalagglomeration of SPIONs with varying degree was observed (FIGS. 8C-8E).The average size measurement of SPIONs with standard deviation for eachsample was calculated. There were two sets of nanoparticles wereobserved in each case (FIG. 8F). The average size of the first set ofparticles was in the range of 3-21 nm and the second of 13-58 nm. Theorder of SPIONs particle size was found to be in the following order:Silicalite>Q-10>Si-SBA-16>MSU-Foam>Si-MCM-41.

Specifically, average SPIONs particle size of the Fe/Si-SBA-16 measuredfrom TEM images was found to be 21.0±1.1 and 9.0±0.3. In supportSi-MCM-41, finely dispersed SPIONs in the range of 3-13 nm was observed.Among the support, silicalite showed larger particles in the range of21-58 nm followed by Q10 silica, which showed of 10-25 nm.

Table 2 shows the adsorption capacity over absence and SPIONs loaded ondifferent nanocarrier supports in solution containing 30 and 60 μg/ml ofcurcumin in 10% methanol-phosphate buffered saline (pH 7) mixture for 24h. The adsorption was measured based on the Beer-Lambert' s law. Theresults show that loading of curcumin over SPIONs impregnated structuredsilica are not affected, rather a slight improvement in the curcuminadsorption was observed compared to parent nanocarriers. Particularly,the percentage adsorption over SiSBA-16 without SPIONs addition was89.1% and 90.0%, while the adsorption capacity after SPIONs loadingimproved to 94.1% and 97.3% with 30 and 60 μg/ml solution, respectively

In the case of Q-10 silica, Si-MCM-41 and silicalite nanocarrier, animprovement of curcumin adsorption over 30 μg/ml solution was observed.

TABLE 2 Adsorption of curcumin over different structured nanocarriers inabsence and . presence of SPIONs in solution containing 30 and 60 μg/mlof curcumin in 10% methanol-phosphate buffered saline (pH 7) mixture for24 h. Metal Initial Final content concentration concentration AdsorptionNanocarrier (wt %) Role (μg/ml) (μg/ml) (%) Q-10 — Single 30 3.18 89.4Fe/Q-10 10 Dual 30 0.64 97.8 Q-10 — Single 60 1.70 97.2 Fe/Q-10 10 Dual60 1.30 97.8 Si-SBA-16 — Single 30 3.25 89.1 Fe/Si-SBA-16 10 Dual 301.76 94.1 Si-SBA-16 — Single 60 6.00 90.0 Fe/Si-SBA-16 10 Dual 60 1.6497.3 Si-MCM-41 — Single 30 2.29 92.4 Fe/Si-MCM-41 10 Dual 30 1.75 94.2Si-MCM-41 — Single 60 2.20 96.3 Fe/Si-MCM-41 10 Dual 60 2.18 96.4Silicalite — Single 30 6.80 78.0 Fe/Silicalite 10 Dual 30 3.25 89.2Silicalite — Single 60 2.50 95.8 Fe/Silicalite 10 Dual 60 2.00 96.6 Thepercentage adsorption was calculated based on the equation

The percentage adsorption was calculated based on the equation:Percentage of curcumin adsorption (%)=(Initial curcumin conc−Finalcurcumin conc)/Initial curcumin conc×100.

The final curcumin concentration was calculated based on the equation=(Final absorbance value×Initial curcumin conc)/Initial absorbancevalue.

FIG. 9 shows the pictorial representation of curcumin adsorption over aSiSBA-16 nanocarrier and SPIONs/SiSBA-16 at different concentrationsranging from 30-390 μg/ml curcumin/nanocarrier in methanol-phosphatebuffered saline (PBS) mixture stirred for 24 h.

The equilibrium adsorption study shows a systematic yellow colorvariation from light yellow to dark yellow occurs over nanocarrierSiSBA-16 (FIG. 9 , samples (a)-(e), while the clear filtrate showseffective adsorption due to large available surface area andaccommodatable pore volume (Table 1). In the case of SPIONs/SiSBA-16,similar effective adsorption was observed with increased curcuminloadings. The filtered solution showed no visible brown colorationindicating no apparent diffusion of adsorbed SPIONs nanoparticles fromsolid to solution phase. Notably, the solid sample coloration afterambient temperature drying showed transformation of color from darkbrown to yellow-brown indicating increased curcumin adsorption; FIG. 9 ,samples (f)-(j).

The curcumin release profile over absence and SPIONs loaded on differentnanocarrier supports (powdered form) in PBS solution (pH 5) for 72 hrare shown in FIGS. 10A-10D. These data show that curcumin delivery ratewas affected by the pore architecture and SPIONs loading of thestructured silica. SPIONs loaded over mesocellular foam showed highestcumulative release than foam itself at fastest rate. The foam composedof largest average pore size (16.4 nm) distribution exhibited highestrelease that reaches 52.3% for 72 h. The relative ease of curcuminrelease with respect to foam type mesosilica shows that the interactionmight be feeble through hydrogen bonding leading to easy cleavage. Itfollows that SPIONs loaded SBA-16 with cubic cage narrow pores (3.3 nm)showed lower but slightly enhanced cumulative release compared to parentSiSBA-16. However, the release trend remains stable over the period of72 h (18.5% for 72 h), which might be due to diffusion through cubictype mesopores. In the case of large type of microspheres Q10 andSPIONs/Q10, showed least curcumin release capability with time (12% in72 h).

In the case of SPIONs/Si-MCM-41 and SPIONs/Silicalite, compared toparent counterpart, a steady cumulative release trend is observed. Thestudy shows that though impregnation of iron oxides produce very lessmagnetically active species, it improves the steadiness of the curcuminrelease behavior. SPIONs/Si-MCM-41 showed cumulative release percentageof 22%, while SPIONs/Silicalite showed 21.1% of curcumin release.Therefore addition of such SPIONs or other types of oxides mayfacilitate steady release. SPIONs loaded over SiKIT-6 and ULPFDU-12showed no appreciable difference in the cumulative release trendcompared to parent SiKIT-6 and ULPFDU-12. SPIONs/SiKIT-6 andSPIONs/ULPFDU-12 showed release of 21.3% and 11.2%, respectively.However, burst release occurs over such nanoformulations. Huang et al.(2012) reported that such release trend are mainly attributed due to thedispersion of drug at the external surface and drug present at theultralarge pore entrance of the 3D channels.

In order to fabricate magnetically active drug delivery system,different structured silicas were evaluated by impregnating constantloading of 10 wt % SPIONs. The X-ray diffraction analysis as shown inFIGS. 1 (a-h) shows that except silicalite, the loaded SPIONs andcurcumin were effectively transformed into amorphous form. The texturalcharacterization (FIG. 2 & Table 1) shows that impregnation of SPIONsover different structured silica has unique textural changes and affectsthe structural integrity of each silica. The spherical Q-10 silica andhexagonal Si-MCM-41 showed very less textural changes with respect tospecific and cumulative surface area over Fe impregnation. Both the typeof silica showed marginal pore filling.

Contrastingly, the cubic 3D Si-SBA-16 with Im3m symmetry and MSU-Foamshowed significant decreases with respect to surface area and as well aspore volume after impregnation. The average pore diameter measurementshows an enlargement after SPIONs impregnation, which signals externaldeposition of Fe₂O₃ particles around the pore walls thereby assistingadditional expanded pores. However, in the case of ULPFDU-12, asignificant loss in the textural changes (both surface area and porevolume) occurs with Fe₂O₃ loadings (Table 1) signaling limited SPIONsloading ability for potential dual applications. The characterization ofmagnetic property shows that Q-10 microsphere silica showed highmagnetic property, followed by SiSBA-16 and mesocellular foam (FIG. 3 ).The diffuse reflectance study shows that octahedral coordinated speciescorresponding to small and large nanoclusters are required to inducemagnetic property (FIG. 4 ). A well dispersed SPIONs in hexagonalstructure and SPIONs present inside the mesopores of SiKIT-6 are tendsto be magnetically non-active (FIG. 3 ). The FT-IR spectroscopy analysisshows that curcumin functionalization over magnetically active supportQ-10 silica and SiSBA-16 occurs majorly inside the structured nanopores,while mesocellular foam indicates presence of certain proportion ofcurcumin at the external surface (FIG. 5 ). SEM (FIGS. 6 & 7 ) and TEMimages of SPIONs over different support (FIG. 8 ) shows the presence ofdifferent types of particle size distributions depending on thestructural features of nanopores. DRS-UV analysis shows the presence ofextra framework species on Q-10, SBA-16 support. While SEM image showsthe uniform characteristics between Q-10 and SBA-16, i.e. microspheres.Both supports has unique characteristics built through microspheresthough with different particle sizes. The presence of such microspheresmight eventually help the Fe₂O₃ nanoparticles to be deposited as extraframework species outside the external surfaces and therefore becomesmagnetically active. Reversely, in case of Si-MCM-41 and Si-KIT-6,nanocarrier, the presence of nanopores with large pore volume (Table 1)helps the SPIONs to be dispersed well throughout the 1D pore channels(as evidenced from TEM image), leading to magnetically inactive species.The formation of finely dispersed SPIONs nanoparticles are confirmedthrough TEM analysis (FIG. 8 ), which showed the nanoparticles are welldispersed in the range of 3-13 nm. In case of Fe/silicalite, extremelylarge sized Fe particles are agglomerated but are not magneticallyactive, which shows that such support might form other form of Fe oxidethat are not magnetically active. The equilibrium adsorption study showsa systematic color variation of SiSBA-16 from yellow to dark yellow,while brown to yellowish brown are observed over SPIONs/SiSBA-16 (FIG. 9).

The preliminary curcumin release profile shows highest cumulativerelease with respect to mesocellular foam, while steady drug releasefound to be for SBA-16. Whereas Q10 silica showed lowest but steadycurcumin release (FIG. 10 ). Comparatively, mesocellular foam showshighest cumulative release within 3 h among the other nanocarriers. TheBET surface area analysis shows that increased pore size distributionfrom 16.4 to 40.2 nm with SPIONs impregnation (FIG. 2 and Table 1).DRS-UV analysis (FIG. 3 ) and FTIR (FIG. 4 ) confirms the presence ofoctahedral Fe₂O₃ species and curcumin at the external surface asnanoclusters. Therefore, the addition of curcumin tends to accommodateat the external surface near the pore mouth and that might be the reasonfor such high cumulative release (Scheme 1). Conversely, in the case ofSiSBA-16, the cumulative release is enhanced with SPIONs addition. Thetextural characterization shows that gradual surface area and porefilling occurs over cubic cage pores without any abrupt pore sizevariations (FIG. 2 and Table 1). The DRS-UV and TEM analysis (FIG. 4 andFIG. 8 ) confirms the presence of octahedral species at the externalsurface as nanoclusters. However, sustained cumulative release trendover SiSBA-16 shows pore diffusional release of curcumin. Such entrappedcurcumin may diffuse through the pores slowly leading to sustainedrelease over the tested drug release period of 72 h.

In case of MSU Foam, curcumin deposition tends to occur at the externalsurface (as evidenced from FT-IR peak of 1028 cm-1) leading to highcumulative release. In the case of hexagonal Si-MCM-41, the presence ofhigh surface area and pore size distribution are able to accommodateSPIONs well as dispersed fine oxides mostly in tetrahedral coordination(FIG. 4 ), which are supported by TEM analysis that shows visible finedispersion of oxides in segregated form (3-13 nm) incorporated well intothe framework rather than desired agglomeration.

Subsequently, the curcumin drug release also reduced with SPIONs loadingthat compete with curcumin. In case of silicalite nanocarrier, anenhancement in the release trend was observed over SPIONs/Silicalite(FIG. 10 ). The cubic cage type of SiKIT-6 (Ia3d symmetry) with thepresence of large surface area and pore volume showed differentaccommodation phenomena. The iron oxides are deposited mostly inside thepore volume of SiKIT-6.

The magnetization analysis showed presence of non-magnetically activespecies over SiKIT-6 (FIG. 3 ). In line with Drs-UV spectroscopy, thoughthe cluster formation occurs which are indicated by the octahedralcoordinated Fe₂O₃ species (FIG. 4 ), the absence of magnetic activespecies shows that the deposition might occurs well inside the cubicpores of KIT-6.

This indicates that despite close textural relation with SBA-16, thedeposition of Fe₂O₃ at the internal or external surface determines themagnetization property, which in turn depends on the unique structuralordering of respective silica.

The pore size distribution of Si-SBA-16 shows the presence of 3D cagetype of mesopores in the range of 5 nm (Table 1) in Im3m symmetry.

The generation of pores using pluronic F127 are reported to producethicker pore walls due to long PO chains of F127, which might help theSPIONs to deposit significant proportional of extra-framework species asnanoclusters.

As shown above nanocarrier textural features are important to tune theiron oxide nanoparticle deposition, which in turn decide the dualresponse for imaging and therapeutics.

As shown by the Example above, the inventors show that SPIONs loaded ondifferent structured nanocarriers provides a multifunctional magneticsilica based nanocarrier loaded with a deliverable curcumin drug. Themagnetic Fe₃O₄ was deposited through enforced impregnation methodologyon the nanostructured pore surfaces followed by calcination, whilecurcumin was functionalized through equilibrium adsorption technique.The VSM analysis showed generation of SPIONs over Q-10, SiSBA-16 andmesocellular foam. The magnetically active support was determined asFe/Q-10>Fe/Si-SBA-16>Fe/MSU-Foam>Fe/Si-MCM-41>Fe/Si-KIT-6. Surface areaanalysis of magnetically active nanocarriers (Q-10, SiSBA-16 andmesocellular foam) showed that pore filling capability and pore sizevariation due to external deposition of SPIONs around the pore walls arerequired to generate magnetically active species. DRS-UV spectroscopyrevealed that hexagonal structure favors uniformly distributed Fe₂O₃ innanosizes, while microspheres Q-10 silica, cubic silica SBA-16 (as shownin SEM images), and mesocellular foam showed agglomerated Fe₂O₃ crystalsas nanoclusters (as evidenced from TEM analysis) and showed superparamagnetic property. ULPFDU-12, KIT-6 and silicalite are found to bemagnetically inactive. The absence and presence of SPIONs over differentnanocarriers were tested for curcumin release for the period of 3 h. Thestudy showed that higher the magnetization, lesser the cumulativerelease capacity of curcumin. The curcumin cumulative release abilityamong magnetically active nanocarriers are as followsMSU-Foam>SiSBA-16>Q-10. In case of magnetically inactive support case,Si-MCM-41 release ability of curcumin reduced but stability of releaseincreased. Silicalite showed improved curcumin release with slightactivity reduction over the period of time. In future, the threesupports can be further scrutinized for multifunctional capability byengineering nanocarrier through silane functionalization, and enhancedcurcumin solubilization technique.

Example 2 Cancer Cell Viability

Materials and Methods/Cell cultures: In this study, a human mammaryadenocarcinoma cell line, MCF7, was used for in vitro testing. MCF7cells were maintained in DMEM (Dulbecco's Modified Eagle Medium) (Gibco,life technologies) supplemented with 10% heat inactivated fetal bovineserum (HI-FBS) (Gibco, life technology), 1% Penicillin Streptomycin(100X-Gibco, life technology), and 1% MEM NEAA (MEM non-essential aminoacids) (100X-Gibco, life technology). Cells were kept in a humidifiedincubator at 37° C. with 5% CO₂. For the experimental setup, MCF7 cellswere seeded on a 96-well plate at a density of 10,000 cells/well. On thenext day, cells were shifted to the starve media (0.5% HI-FBS containingmedia) for 24 h before treatment.

Treatment: Six groups were tested: Mesocellular foam silica (group I),Fe₂O₃ (group II), curcumin (group III), Mesocellular foam silica+Fe₂O₃(group IV), silica+curcumin (group V), and Mesocellular foamsilica+Fe₂O₃+curcumin (group VI). A stock solution of each condition wasfreshly prepared for every experiment using 1×PBS (Gibco, lifetechnology) as a vehicle. Subsequently, cells were treated withincreasing concentrations of each group as follows: 10, 20, 40, 80, and100 μg/ml for 24 h.

Cell viability MTT Assay: The viability of cells was tested using3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)assay. It is based on the ability to reduce MTT to formazan crystals.The assay was performed using previously published protocols (Mosmann T.Rapid colorimetric assay for cellular growth and survival: applicationto proliferation and cytotoxicity assays. J Immunol Methods. 1983 Dec.16; 65(1-2):55-63. PubMed PMID: 6606682). Briefly, MTT (Sigma-Aldrich)was dissolved in PBS at 5 mg/ml. Working solution of MTT was prepared ata final concentration of 0.5 mg/ml (10 μl of stock MTT+90 μl1×PBS/well). The 96-well plate was washed twice with 1×PBS and 100 μl ofMTT working solution was dispended in all wells. An MTT backgroundcontrol was included, in which MTT working solution was added to emptywells (i.e. no cells). The plate was incubated for three hours at 37°C., followed by the addition of 100 μl of acidified isopropanolsolubilizing solution (0.04N HCL isopropanol). The change in colorintensity was measured at 570 nm wavelength using SYNERGY-neo2 BioTekELISA reader. Each condition was performed in triplicates. The readingof each triplicate was averaged and subtracted from the averaged MTTbackground control reading. Each condition was compared to the control(no treatment) wells. The following equation was used to calculate the %of cell viability:

${\%{Cell}{Viability}} = {\frac{{averaged}{sample}{read}}{{averaged}{control}{read}} \times 100}$

Statistics: Cell viability assay data represent five independentexperiments. Statistical analysis was performed using Prism 7 software(GraphPad). Analysis was performed using one-way ANOVA with Dunnett'spost hoc test.

To investigate the cytotoxic effects of curcumin-loaded/Fe₂O₃impregnated mesocellular foam silica nanoparticles, the inventorsassessed cell viability using the MTT assay on MCF7 cells. In thatassay, healthy cells will be able to reduce MTT to the purple-coloredformazan, while unhealthy/dead cells cannot. MCF7 cells were treatedwith the following conditions: mesocellular foam silica (group I), Fe₂O₃(group II), curcumin (group III), mesocellular foam silica+Fe₂O₃ (groupIV), mesocellular foam silica+curcumin (group V), and mesocellular foamsilica+Fe₂O₃+curcumin (group VI) at increasing concentrations (10, 20,40, 80, and 100 μl g/ml) for 24 h (FIG. 11 ).

Mesocellular foam silica and Fe₂O₃ did not elicit any effect on cellviability either individually (groups I, II) or when combined (groupIV). However, curcumin significantly reduced cell viability on its own(group III) and when combined with others (groups V, VI).

Curcumin alone (group III) was able to reduce cell viability to 66.6%and maintain that reduction throughout the different concentrations.Interestingly, when curcumin was combined with either mesocellular foamsilica or mesocellular foam silica and Fe₂O₃ (groups V, VI), it had adose dependent reduction in viability that reached to 48.9% (at 100μg/ml) and 55.4% (at 50 μg/ml), respectively.

It is worth mentioning that when preparing curcumin stock solutions, 390μg/ml was used for groups III, while only 6.12 μg/ml of curcuminadsorbed on the mesosilica nanoparticles was used for V and VI. Thismight explain why groups V and VI had a gradual reduction in viability,while group III did not. It also emphasizes the higher efficiency ofcurcumin when encapsulated in these mesosilica nanoparticles. Theseresults show that the Fe₂O₃-coated silica nanoparticles that are loadedwith curcumin can effectively reduce viability of the human breastcancer cell line, MCF7 and the therapeutic and drug-delivery advantagesof the invention.

As shown in FIG. 11 , Fe₂O₃ impregnated silica nanoparticles loaded withcurcumin significantly reduced cell viability. Percentage of cellviability with the following treatments: mesocellular foam silica,Fe₂O₃, curcumin, mesocellular foam silica+Fe₂O₃, mesocellular foamsilica+curcumin, and mesocellular foam silica+Fe₂O₃+ curcumin. Treatmentconcentrations were: 10, 20, 40, 80, and 100 μg/ml for 24 h (n=5independent experiments). The dashed line represents control which wasset as 100% cell viability. Error bars, ±SEM. *P<0.05; **P<0.01 versuscontrol.

Using the MTT cell viability assay, these results show that curcuminindeed reduced cell viability. While 390 μg/ml of curcumin was used tobe adsorbed over mesosilica, the equilibrium adsorption showed thepresence of 6.12 μg/ml in SPIONs/meso mesocellular foam silicananoformulation. These in vitro experiments compared curcumin alone(group III—390 μg/ml) to the equilibrium-adsorbedcurcumin/SPIONS/mesocellular foam nanoformulation (groups V and VI—6.12μg/ml). The study showed that curcumin/SPIONs/mesocellular foam silicacomposite with very low concentration of 6.12 μg/ml was very effectiveto exert a cytotoxic effect on the breast cancer cell line MCF7. It isabout 65 times lower than the required concentration for curcumin alone.This shows the high bioavailability of nanoformulation.

Terminology. Terminology used herein is for the purpose of describingparticular embodiments only and is not intended to be limiting of theinvention.

The headings (such as “Background” and “Summary”) and sub-headings usedherein are intended only for general organization of topics within thepresent invention, and are not intended to limit the disclosure of thepresent invention or any aspect thereof. In particular, subject matterdisclosed in the “Background” may include novel technology and may notconstitute a recitation of prior art. Subject matter disclosed in the“Summary” is not an exhaustive or complete disclosure of the entirescope of the technology or any embodiments thereof. Classification ordiscussion of a material within a section of this specification ashaving a particular utility is made for convenience, and no inferenceshould be drawn that the material must necessarily or solely function inaccordance with its classification herein when it is used in any givencomposition.

As used herein, the singular forms “a”, “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise.

It will be further understood that the terms “comprises” and/or“comprising,” when used in this specification, specify the presence ofstated features, steps, operations, elements, and/or components, but donot preclude the presence or addition of one or more other features,steps, operations, elements, components, and/or groups thereof.

As used herein, the term “and/or” includes any and all combinations ofone or more of the associated listed items and may be abbreviated as“/”.

As used herein in the specification and claims, including as used in theexamples and unless otherwise expressly specified, all numbers may beread as if prefaced by the word “substantially”, “about” or“approximately,” even if the term does not expressly appear. The phrase“about” or “approximately” may be used when describing magnitude and/orposition to indicate that the value and/or position described is withina reasonable expected range of values and/or positions. For example, anumeric value may have a value that is +/−0.1% of the stated value (orrange of values), +/−1% of the stated value (or range of values), +/−2%of the stated value (or range of values), +/−5% of the stated value (orrange of values), +/−10% of the stated value (or range of values),+/−15% of the stated value (or range of values), +/−20% of the statedvalue (or range of values), etc. Any numerical range recited herein isintended to include all sub-ranges subsumed therein.

Disclosure of values and ranges of values for specific parameters (suchas temperatures, molecular weights, weight percentages, etc.) are notexclusive of other values and ranges of values useful herein. It isenvisioned that two or more specific exemplified values for a givenparameter may define endpoints for a range of values that may be claimedfor the parameter. For example, if Parameter X is exemplified herein tohave value A and also exemplified to have value Z, it is envisioned thatparameter X may have a range of values from about A to about Z.Similarly, it is envisioned that disclosure of two or more ranges ofvalues for a parameter (whether such ranges are nested, overlapping ordistinct) subsume all possible combination of ranges for the value thatmight be claimed using endpoints of the disclosed ranges. For example,if parameter X is exemplified herein to have values in the range of 1-10it also describes subranges for Parameter X including 1-9, 1-8, 1-7,2-9, 2-8, 2-7, 3-9, 3-8, 3-7, 2-8, 3-7, 4-6, or 7-10, 8-10 or 9-10 asmere examples. A range encompasses its endpoints as well as valuesinside of an endpoint, for example, the range 0-5 includes 0, >0, 1, 2,3, 4, <5 and 5.

As used herein, the words “preferred” and “preferably” refer toembodiments of the technology that afford certain benefits, undercertain circumstances. However, other embodiments may also be preferred,under the same or other circumstances. Furthermore, the recitation ofone or more preferred embodiments does not imply that other embodimentsare not useful, and is not intended to exclude other embodiments fromthe scope of the technology. As referred to herein, all compositionalpercentages are by weight of the total composition, unless otherwisespecified. As used herein, the word “include,” and its variants, isintended to be non-limiting, such that recitation of items in a list isnot to the exclusion of other like items that may also be useful in thematerials, compositions, devices, and methods of this technology.Similarly, the terms “can” and “may” and their variants are intended tobe non-limiting, such that recitation that an embodiment can or maycomprise certain elements or features does not exclude other embodimentsof the present invention that do not contain those elements or features.

The description and specific examples, while indicating embodiments ofthe technology, are intended for purposes of illustration only and arenot intended to limit the scope of the technology. Moreover, recitationof multiple embodiments having stated features is not intended toexclude other embodiments having additional features, or otherembodiments incorporating different combinations of the stated features.Specific examples are provided for illustrative purposes of how to makeand use the compositions and methods of this technology and, unlessexplicitly stated otherwise, are not intended to be a representationthat given embodiments of this technology have, or have not, been madeor tested.

All publications and patent applications mentioned in this specificationare herein incorporated by reference in their entirety to the sameextent as if each individual publication or patent application wasspecifically and individually indicated to be incorporated by reference,especially referenced is disclosure appearing in the same sentence,paragraph, page or section of the specification in which theincorporation by reference appears.

The citation of references herein does not constitute an admission thatthose references are prior art or have any relevance to thepatentability of the technology disclosed herein. Any discussion of thecontent of references cited is intended merely to provide a generalsummary of assertions made by the authors of the references, and doesnot constitute an admission as to the accuracy of the content of suchreferences.

The invention claimed is:
 1. An equilibrium adsorption method for makinga silica nanocarrier comprising superparamagnetic iron oxidenanoparticles and curcumin, comprising: impregnating nanostructured poresurfaces of a platform of nanoporous structured silica withsuperparamagnetic iron oxide nanoparticles (SPIONs) to form animpregnated platform of structured silica, calcining the impregnatedplatform of structured silica between 500 and 900° C. to form a calcinedplatform of structured silica, and adsorbing a curcuminoid onto thecalcined platform of structure silica and magnetic nanoparticles to formthe silica nanocarrier, wherein the adsorbing includes mixing thecalcined platform of structure silica and magnetic nanoparticles with anaqueous solution of the curcuminoid, wherein the silica nanocarriercomprises a platform of nanoporous structured silica selected from thegroup consisting of SiSBA-16, Q-10 silica, mesocellular foam,silicalite, mesosilicalite, SiKIT-6, ULPFDU-12, SiMCM-41, ZSM-5, USY,Mordenite, ZSM-11, ZSM-12, ZSM-22, ZSM-23, mesocarbon, graphene oxideand mixtures thereof, wherein the superparamagnetic iron oxidenanoparticles (SPIONS) are present in an amount ranging from about 5 wt% to about 30 wt % based on total weight of the silica nanocarrier, andwherein the curcuminoid is adsorbed on the surface of the nanoporousstructured silica.
 2. The equilibrium adsorption method of claim 1,wherein the platform of nanoporous structured silica is at least one ofMSU-foam, SiSBA-16, or Q-10; and the curcuminoid is curcumin.
 3. Theequilibrium adsorption method of claim 1, wherein the platform ofnanoporous structured silica comprises MSU-foam.
 4. The equilibriumadsorption method of claim 1, wherein the platform of nanoporousstructured silica comprises SiSBA-16.
 5. The equilibrium adsorptionmethod of claim 1, wherein the platform of nanoporous structured silicacomprises Q-10.
 6. The equilibrium adsorption method of claim 1, whereinthe SPIONs comprise Fe₂O₃ or a mixture of NiFe₂O₄, CuFe₂O₄, MnFe₂O₄ orCoFe₂O₄.
 7. The equilibrium adsorption method of claim 1, wherein theSPIONs comprise γ-Fe₂O₃.
 8. The equilibrium adsorption method of claim1, wherein the SPIONs have an average particle size ranging from about 7to about 18 nm when the platform of nanoporous structured silica isMSU-foam; about 9 to 21 nm when the platform of nanoporous structuredsilica is SiSBA-16; or about 10 to about 25 nm when the platform ofnanoporous structured silica is Q-10.
 9. The equilibrium adsorptionmethod of claim 1, wherein the curcuminoid is present in an amountranging from 50 to 70 wt %.
 10. The equilibrium adsorption method ofclaim 1, wherein the silica nanocarrier further comprises a polymer,wherein the SPIONs and/or the curcuminoid is covered with orincorporated into the polymer; and/or wherein one or more components ofthe composition is functionalized with chitosan, polyacrylic acid, PLGA,or another agent to increase its biocompatibility in vivo.
 11. Theequilibrium adsorption method of claim 1, wherein the silica nanocarrierfurther comprises at least one antibody or other targeting agent thatbinds to cancer cells, neoplasm cells, or tumor cells.
 12. Theequilibrium adsorption method of claim 1, wherein the platform ofnanoporous structured silica consists of SiSBA-16, Q-10 silica,mesocellular foam, silicalite, mesosilicalite, SiKIT-6, ULPFDU-12 orSiMCM-41.